Recovery of higher alcohols from dilute aqueous solutions

ABSTRACT

This invention is directed to methods for recovery of C3-C6 alcohols from dilute aqueous solutions, such as fermentation broths. Such methods provide improved volumetric productivity for the fermentation and allows recovery of the alcohol. Such methods also allow for reduced energy use in the production and drying of spent fermentation broth due to increased effective concentration of the alcohol product by the simultaneous fermentation and recovery process which increases the quantity of alcohol produced and recovered per quantity of fermentation broth dried. Thus, the invention allows for production and recovery of C3-C6 alcohols at low capital and reduced operating costs.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/342,992, filed Dec. 23, 2008, which claims priority to U.S.Provisional Application Ser. No. 61/017,141 filed Dec. 27, 2007;61/021,495 filed Jan. 16, 2008; 61/021,558 filed Jan. 16, 2008,61/021,567 filed Jan. 16, 2008, and 61/016,483 filed Dec. 23, 2007.Accordingly, this application incorporates by reference in its entiretyall subject matter of each of U.S. Provisional Application Ser. No.61/017,141 filed Dec. 27, 2007; 61/021,495 filed Jan. 16, 2008;61/021,558 filed Jan. 16, 2008; and 61/021,567 filed Jan. 6, 2008 forall purposes.

FIELD OF THE INVENTION

This application relates generally to methods for recovery of C3-C6alcohols from dilute aqueous solutions, such as fermentation broths.

BACKGROUND OF THE INVENTION

Biofuels have a long history ranging back to the beginning of the 20thcentury. As early as 1900, Rudolf Diesel demonstrated at the WorldExhibition in Paris, France, an engine running on peanut oil. Soonthereafter, Henry Ford demonstrated his Model T running on ethanolderived from corn. Petroleum-derived fuels displaced biofuels in the1930s and 1940s due to increased supply, and efficiency at a lower cost.

Market fluctuations in the 1970s, due the Arab oil embargo and theIranian revolution, coupled to the decrease in US oil production, led toan increase in crude oil prices and a renewed interest in biofuels.Today, many interest groups, including policy makers, industry planners,aware citizens, and the financial community, are interested insubstituting petroleum-derived fuels with biomass-derived biofuels. Theleading motivation for developing biofuels is of economical nature,namely, the threat of ‘peak oil’, the point at which the consumptionrate of crude oil exceeds the supply rate, thus leading to significantlyincreased fuel cost results in an increased demand for alternativefuels.

Biofuels tend to be produced with local agricultural resources in many,relatively small facilities, and are seen as a stable and secure supplyof fuels independent of geopolitical problems associated with petroleum.At the same time, biofuels enhance the agricultural sector of nationaleconomies. In addition, since fossil sources of fuels take hundreds ofmillions of years to be regenerated and their use increases carbondioxide levels in the atmosphere, leading to climate change concerns,sustainability is an important social and ethical driving force which isstarting to result in government regulations and policies such as capson carbon dioxide emissions from automobiles, taxes on carbon dioxideemissions, and tax incentives for the use of biofuels.

The acceptance of biofuels depends primarily on economicalcompetitiveness of biofuels when compared to petroleum-derived fuels.Biofuels that cannot compete in cost with petroleum-derived fuels willbe limited to specialty applications and niche markets. Today, the useof biofuels is limited to ethanol and biodiesel. Currently, ethanol ismade by fermentation from corn in the US, sugar cane in Brazil, andother grains worldwide. Ethanol is competitive with petroleum-derivedgasoline, exclusive of subsidies or tax benefits, if crude oil staysabove $50 per barrel. Biodiesel has a breakeven price of crude oil ofover $60/barrel to be competitive with petroleum-based diesel (NexantChem Systems, 2006, Final Report, Liquid Biofuels: Substituting forPetroleum, White Plains, N.Y.).

Several factors influence the core operating costs of a carbohydratebased biofuel source. In addition to the cost of the carbon-containing,plant produced raw material, a key factor in product economic costs forethanol or other potential alcohol based biofuels, such as butanol, isthe recovery and purification of biofuels from aqueous streams. Manytechnical approaches have been developed for the economic removal ofalcohols from aqueous based fermentation media. The most widely usedrecovery techniques today use distillation and molecular sieve drying toproduce ethanol. For example, butanol production via theClostridia-based acetone-butanol-ethanol fermentation also relied ondistillation for recovery and purification of the products. Distillationfrom aqueous solutions is energy intensive. For ethanol, additionalprocessing equipment to break the ethanol/water azeotrope is required.This equipment, molecular sieves, also uses significant quantities ofenergy.

Many unit operations have been studied for the recovery and purificationof fermentation produced alcohols, including filtration, liquid/liquidextraction, membrane separations (e.g., tangential flow filtration,pervaporation, and perstraction), gas stripping, and “salting out” ofsolution, adsorption, and absorption. Each of the approaches hasadvantages and disadvantages depending on the circumstances of theproduct to be recovered and the product's physical and chemicalproperties and the matrix in which it resides.

Variables which control the production costs of biofuels can becharacterized as those impacting operating costs, capital costs, orboth. Typically, key variables that control fermentation economicperformance include carbohydrate yield to desired product, productconcentration and volumetric productivity. All three key variables,yield, product concentration, and volumetric productivity, impact bothcapital and operating costs.

As product yield on carbohydrate fermented is increased, the productioncosts for a given unit of product decrease linearly relative to rawmaterial costs. The product yield on carbohydrate also impacts equipmentsize, capital expenditures, utilities consumption and feed stockpreparation materials such as enzymes, minerals, nutrients (vitamins),and water. For example an increase in product yield on glucose tobutanol from 50% to 90% of theoretical results in a 44% decrease indirect operating costs. Also, the increased yield of 90% reduces theamount of raw materials handled and processed. The increased yielddirectly reduces capital investment required for the production facilityas all equipment from carbohydrate preparation through purification andrecovery are reduced in size. Equipment, piping, and utilityrequirements can be reduced by 32% if yield is increased from 50% to90%. The direct influence of product yield on production costs makes ita key influence on the cost and market viability for biofuels. Anapproach to increase product yield involves Genetically EngineeredMicroorganisms (GEMs) that can be constructed to manipulate theorganism's metabolic pathway to reduce or eliminate undesired products,increase the efficiency of the desired metabolite or both. This allowsfor the deletion of one or both of low cost products and undesiredproducts, which increases production of desired products.

For example, US Patent Application Publication 2005/0089979 discloses afermentation process that utilizes a Clostridium beijerinckiimicroorganism that produces a mixture of products including 5.3 g/Lacetone, 11.8 g/L butanol, and 0.5 g/L ethanol. An appropriatelymodified Genetically Engineered Microorganism eliminates acetone andethanol production while increasing conversion of carbohydrates tobutanol. The redirection of a carbohydrate feedstock away from ethanoland acetone to butanol increases butanol production from 11.8 g/L to18.9 g/L, a 60% increase in butanol production relative to carbohydrateconsumption. The elimination of the ethanol and acetone byproducts alsoallows for reduced capital costs as less equipment is necessary tocomplete recovery and purification.

Application of biochemical tools, including, genetic engineering andclassical strain development can also impact the final productconcentration (g/L) and fermentation volumetric productivity (g/L-hr) ofthe biocatalyst. Final product concentration and volumetric productivityimpacts several aspects of product economics, including equipment size,raw material use, and utility costs. As the tolerable productconcentration increases in the fermentation, recovery volumes of aqueoussolutions are decreased which results in reduced capital costs andsmaller volumes of materials to process within the production facility.

Volumetric productivity directly impacts the required fermentor capacityto achieve the same product output. For example, a traditionalClostridium beijerinckii acetone-butanol-ethanol (ABE) fermentationproduces a ratio of acetone, butanol, and ethanol. Geneticallyengineered microbes allow the designed production of a single product,such as n-butanol, isobutanol or 2-butanol (Donaldson et al., U.S.patent application Ser. No. 11/586,315). Butanol tolerant hosts can beidentified utilizing techniques to identify and enhance the butanoltolerance (Bramucci et al., U.S. patent application Ser. No.11/743,220). These two techniques can then be combined to producebutanol at commercially relative concentrations, and volumetricproductivity.

The utilization of GEMs to increase product volumetric productivity andconcentration may strongly influence product economics. For example, abutanol fermentation completed at twice the volumetric productivity willreduce fermentor cost by almost 50% for a large industrial biofuelsfermentation facility. The fermentor capital cost and size reductiondecreases depreciation and operating costs for the facility. Similarly,if the GEMs result in an organism that is tolerant to higher butanolconcentrations, operating and capital costs are reduced for a givenproduction volume. For example, if a wild type strain is capable oftolerating 20 g/L butanol and a corresponding genetically improved orgenetically enhanced microorganism tolerates 40 g/L butanol, the waterload in the fermentor broth volume handled in downstream recovery andpurification equipment is reduced by half. In this example, the doublingof product concentration in the fermentation broth almost halves theamount of water to be recovered and processed in recovery unitoperations.

A large number of minor cost components also impact operating andcapital costs for biofuels production. Example factors that can impactfermentation include, but are not limited to, chemical additives, pHcontrol, surfactants, and contamination are some of the factors but manyadditional factors can impact fermentation product cost.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a method to recover aC3-C6 alcohol from a fermentation broth containing microorganisms andthe C3-C6 alcohol. The method includes the step of increasing theactivity of the C3-C6 alcohol in a portion of the fermentation broth toat least that of saturation of the C3-C6 alcohol in the portion.Alternatively, the method may include the step of decreasing theactivity of water in a portion of the fermentation broth to at leastthat of saturation of the C3-C6 alcohol in the portion. The method alsoincludes the step of forming a C3-C6 alcohol-rich liquid phase and awater-rich liquid phase from the portion of the fermentation broth. Themethod also includes the step of separating the C3-C6 alcohol-rich phasefrom the water-rich phase.

In another embodiment, the present invention provides a method toproduce a C3-C6 alcohol. The method includes the step of culturing amicroorganism in a fermentation medium to produce the C3-C6 alcohol. Themethod also includes the step of increasing the activity of the C3-C6alcohol in a portion of the fermentation medium to at least that ofsaturation of the C3-C6 alcohol in the fermentation medium. The methodalso includes the step of forming a C3-C6 alcohol-rich liquid phase anda water-rich phase from the portion of the fermentation medium. Themethod also includes the step of separating the C3-C6 alcohol-rich phasefrom the water-rich phase. The method also includes the step ofconducting the water rich phase to the fermentation medium.

In another embodiment, the present invention provides a method toproduce a C3-C6 alcohol. The method includes hydrolyzing a feedstockcomprising a polysaccharide and at least one other compound to producefermentable hydrolysis products. The method also includes fermenting atleast a portion of the fermentable hydrolysis products in a fermentationmedium to produce the C3-C6 alcohol, wherein the fermentation mediumfurther includes at least one non-fermented compound. The method alsoincludes increasing the activity of the C3-C6 alcohol from a portion ofthe fermentation medium to at least that of saturation of the C3-C6alcohol in the fermentation medium. The method also includes forming aC3-C6 alcohol-rich liquid phase and a water-rich phase from the portionof the fermentation medium. The method also includes separating theC3-C6 alcohol-rich phase from the water-rich phase. The method alsoincludes separating the at least one non-fermented compound from thefermentation medium, the water-rich phase or both. In some embodiments,the step of hydrolyzing a feedstock includes saccharification. In someembodiments, at least two of the steps of hydrolyzing, fermenting andincreasing the activity of the C3-C6 alcohol are conductedsimultaneously for at least a portion of time. In some embodiments, thestep of fermenting is conducted with a microorganism that is capable ofhydrolyzing the feedstock.

In another embodiment, the present invention provides a method toproduce a product from a C3-C6 alcohol in a fermentation broth includingmicroorganisms and the C3-C6 alcohol. The method includes the steps ofdistilling a vapor phase comprising water and C3-C6 alcohol from thefermentation broth and reacting the C3-C6 alcohol in the vapor phase toform the product. In some embodiments, the C3-C6 alcohol is propanol andthe ratio of the C3-C6 alcohol to water in the vapor phase is greaterthan about 0.2 (w/w). In some embodiments, the C3-C6 alcohol is butanoland the ratio of the C3-C6 alcohol to water in the vapor phase isgreater than about 1 (w/w). In some embodiments, the C3-C6 alcohol ispentanol and the ratio of the C3-C6 alcohol to water in the vapor phaseis greater than about 4 (w/w). In some embodiments, the step of reactingis in the presence of a catalyst. In some embodiments, the catalyst is aheterogeneous catalyst.

In another embodiment, the present invention provides a method toproduce a C3-C6 alcohol. This method includes the steps of culturing amicroorganism in a fermentation medium to produce the C3-C6 alcohol.This method also includes the step of increasing the activity of theC3-C6 alcohol in a portion of the fermentation medium. The method alsoincludes the step of distilling the portion of the fermentation mediumto produce a vapor phase that includes water and C3-C6 alcohol and aliquid phase, and the step of conducting the liquid phase to thefermentation medium.

In another embodiment, the present invention provides a method torecover a C3-C6 alcohol from a dilute aqueous solution that includes afirst amount of the C3-C6 alcohol. The method includes distilling aportion of the dilute aqueous solution to a vapor phase containing C3-C6alcohol and water, wherein the vapor phase contains between about 1% byweight and about 45% by weight of the first amount of C3-C6 alcohol fromthe portion of the dilute aqueous solution. The method further includescondensing the vapor phase. In various embodiments, the vapor phase maycontain between about 2% by weight and about 40%, between about 3% byweight and about 35%, between about 4% by weight and about 30%, orbetween about 5% by weight and about 25% by weight of the C3-C6 alcoholfrom the dilute aqueous solution. In some embodiments, the methodincludes forming a C3-C6 alcohol-rich liquid phase and a water-richliquid phase from the condensed vapor phase. In some embodiments, themethod includes separating the C3-C6 alcohol-rich phase and thewater-rich phase. In some embodiments, the step of distilling is asingle stage distillation. The distilling may be adiabatic orisothermal. In various embodiments, the enrichment of alcohol from thedilute aqueous solution to the condensed vapor may be at least about 5fold, at least about 10 fold or at least about 15 fold.

In another embodiment, the present invention provides a method tooperate a retrofit ethanol production plant. In some embodiments, theretrofit plant includes a pretreatment unit, multiple fermentationunits, and a beer still to produce a C3-C6 alcohol. The method includespretreating a feedstock to form fermentable sugars in the pretreatmentunit. The method also includes culturing a microorganism in afermentation medium comprising the fermentable sugars in a firstfermentation unit to produce the C3-C6 alcohol. The method also includestreating a portion of the fermentation medium comprising the C3-C6alcohol to remove a portion of the C3-C6 alcohol. The method alsoincludes returning the treated portion of the fermentation medium to thefirst fermentation unit. The method also includes transferring thefermentation medium from the first fermentation unit to the beer still.In various embodiments, the C3-C6 alcohol output of the retrofit plantis at least about 80%, at least about 90%, or at least about 95% of theC3-C6 alcohol equivalent of the ethanol maximum output of the plantbefore retrofit.

In another embodiment, the present invention provides a method forextraction of a C3-C6 alcohol from an aqueous solution. The methodincludes contacting the aqueous solution with an acidic, amine-basedalcohol-selective extractant. In some embodiments, two phases form afterthe step of contacting. In some embodiments, the acidic amine basedextractant is formed by acidifying an organic amine solution.

In various embodiments, the ratio of the C3-C6 alcohol to water in thefermentation broth or fermentation medium may be less than about 9/91(w/w), less than about 6/94 (w/w) or less than about 3/97 (w/w).

In some embodiments, the step of increasing the activity may includeaddition of a hydrophilic solute. In some embodiments, this step mayinclude distilling a vapor phase containing water and C3-C6 alcohol. Insome embodiments, this step may include reverse osmosis. In someembodiments, this step may include dialysis. In some embodiments, thisstep may include adsorption of the C3-C6 alcohol on an alcohol-selectiveadsorbent. In some embodiments, this step may include extraction of theC3-C6 alcohol into an alcohol-selective extractant. In some embodiments,this step may include adsorption of water on a water-selectiveadsorbent. In some embodiments, this step may include extraction ofwater into a water-selective extractant. In some embodiments, this stepmay include selective removal of water, selective binding of water, orselective rejection of water.

In various embodiments, the C3-C6 alcohol is propanol, butanol,pentanol, or hexanol. In some embodiments, the propanol may be1-propanol or 2-propanol. In some embodiments, the butanol may be1-butanol, 2-butanol, tert-butanol (2-methyl-2 propanol), or iso-butanol(2-methyl-1-propanol). In some embodiments, the pentanol may be1-pentanol, 2-pentanol, 3-10 pentanol, 2-methyl-1-butanol,3-methyl-1-butanol, 2-methyl-2-butanol, 3-methyl-2-butanol, or2,2-dimethyl-1-propanol. In some embodiments, the hexanol may be1-hexanol, 2-hexanol, 3-hexanol, 2-methyl-1-pentanol,3-methyl-1-pentanol, 4-methyl-1-pentanol, 2-methyl-2-pentanol,3-methyl-2-pentanol, 4-methyl-2 pentanol, 2-methyl-3-pentanol,3-methyl-3-pentanol, 3,3-dimethyl-1-butanol, 2,2-dimethyl-1-butanol,2,3-dimethyl-1-butanol, 2,3-dimethyl-2-butanol, 3,3-dimethyl-2-butanol,or 2 ethyl-1-butanol. In a preferred embodiment, the C3-C6 alcohol isiso-butanol.

In some embodiments, the method of the instant invention furtherincludes the step of cooling the alcohol-rich phase to increase theratio of the alcohol to water in the alcohol-rich phase.

In some embodiments, the method includes the step of recovering theC3-C6 alcohol from the alcohol-rich phase. In various embodiments, thestep of recovering may include the step of distillation, dialysis, wateradsorption, extraction of the C3-C6 alcohol by solvent extraction,contact with a hydrocarbon liquid that is immiscible in water or contactwith a hydrophilic compound. This step may produce two phases includinga first phase containing the C3-C6 alcohol and water and a second phasecontaining the C3-C6 alcohol, wherein the ratio of water to C3-C6alcohol in the second phase is less than in the first phase. In variousembodiments, the second phase may contain at least about 90% by weightalcohol, at least about 95% by weight alcohol or at least about 99% byweight alcohol.

In some embodiments, the step of recovering may include distilling theC3-C6 alcohol-rich phase. In such embodiments, the first phase is avapor phase containing the alcohol and water and the second phase is ahigh boiling product containing the alcohol. In some embodiments, themethod may further include combining the fermentation broth with thehigh boiling product, prior to the step of increasing the activity ofthe C3-C6 alcohol. In some embodiments, the method may include combiningthe fermentation broth with the water-rich phase prior to the step ofincreasing the activity of the C3-C6 alcohol.

In some embodiments, after the step of increasing the activity, theremaining portion of the dilute aqueous solution may be conducted to afermentation vessel. In some embodiments, the remaining portion maycontain an impurity and the method further includes removing at least aportion of the impurity from at least a portion of the remaining portionbefore conducting the solution to the fermentation vessel. In someembodiments, the fermentation broth contains an impurity and the ratioof the impurity to the C3-C6 alcohol in the C3-C6 alcohol-rich liquidphase is greater than the ratio in the water-rich phase.

In some embodiments, the step of increasing the activity of the C3-C6alcohol may include distilling a vapor phase containing water andalcohol and condensing the vapor phase. In some of these embodiments,the portion of the fermentation broth may be treated for water removalbefore the step of distilling. In some embodiments, the C3-C6alcohol-rich phase may be treated for water removal. In someembodiments, water removal may be done simultaneously with the step ofdistilling. The water removal may be by selective removal of water,selective binding of water or selective rejection of water. In variousembodiments, the water removal may be effected by addition of ahydrophilic solute, addition of a carbon source, reverse osmosis,dialysis, adsorption of the alcohol on an alcohol-selective adsorbent,extraction of the alcohol into an alcohol-selective extractant,adsorption of water on a water-selective adsorbent or extraction ofwater into a water selective extractant.

In some embodiments, the ratio of the C3-C6 alcohol to water in theC3-C6 alcohol-rich phase is greater than the ratio of the C3-C6 alcoholto water in the fermentation broth by at least about 5 fold.

In some embodiments, distilling is conducted at below atmosphericpressure and at a temperature of between about 20° C. and about 95° C.In some embodiments, the step of distilling is conducted at a pressureof from about 0.025 bar to about 10 bar. The step of distilling may beconducted in a fermentation vessel or in a distillation vessel. In someembodiments, the portion of the fermentation broth is at the temperatureof between about 20° C. and about 95° C. prior to introduction into thedistillation vessel. In some embodiments, the portion of thefermentation broth is subjected to below atmospheric pressure in thedistillation vessel. In some embodiments, after the step of distilling,the remaining portion of the fermentation broth is conducted from thedistillation vessel to the fermentation vessel. In various embodiments,the fermentation vessel may be at the atmospheric pressure, belowatmospheric pressure or above atmospheric pressure.

In some embodiments, the C3-C6 alcohol is propanol and the ratio of thealcohol to water in the alcohol-rich phase is greater than about 0.2(w/w). In some embodiments, the C3-C6 alcohol is butanol and the ratioof the alcohol to water in the alcohol-rich phase is greater than about1 (w/w). In some embodiments, the C3-C6 alcohol is pentanol and theratio of the C3-C6 alcohol to water in the C3-C6 alcohol-rich phase isgreater than about 4 (w/w).

In some embodiments, where the step of increasing activity of thealcohol includes distilling a vapor phase containing water and the C3-C6alcohol and condensing the vapor phase, the vapor phase may contain anazeotrope of water and the alcohol. In some embodiments, the ratio ofthe alcohol to water in the alcohol-rich phase is greater than the ratioin the azeotrope.

In some embodiments, the methods of the present invention furtherinclude processing the C3-C6 alcohol-rich phase. In some embodiments,the step of processing may include distilling substantially pure C3-C6alcohol from the C3-C6 alcohol-rich phase. In some embodiments,processing may include distilling an azeotrope of the C3-C6 alcohol fromthe C3-C6 alcohol-rich phase. In some embodiments, processing mayinclude contacting the C3-C6 alcohol-rich phase with a C3-C6alcohol-selective adsorbent. In some embodiments, processing may includeconverting C3-C6 alcohol in the C3-C6 alcohol-rich phase to an olefin.In some embodiments, processing may include combining the C3-C6alcohol-rich phase with a hydrocarbon liquid that is immiscible inwater. In some embodiments, the combination may form a single uniformphase. In some embodiments, the combination may form a light phase and aheavy phase and the ratio of alcohol to water in the light phase may begreater than the ratio in the heavy phase.

In some embodiments, the culturing may include the process of batchfermentation, a fed-batch fermentation, continuous fermentation, cellrecycle fermentation, or an enzyme reaction process. In someembodiments, the microorganism may be Clostridium butyricum, Clostridiumacetobutylicum, Clostridium saccharoperbutylacetonicum, Clostridiumsaccharobutylicum or Clostridium beijerinckii. In some embodiments, themicroorganism may be a temperature resistant microorganism. In someembodiments, the microorganism may be viable at temperatures from about20° C. to about 95° C. In some embodiments, the microorganism may have aproductivity of at least about 0.5 g/L per hour.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents an embodiment of the present invention for theproduction and recovery of iso-butanol.

FIG. 2 represents an embodiment of the present invention for theproduction and recovery of butanol from fermentation broth in a processof simultaneous saccharification and fermentation of pretreated corn.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes methods for recovery of C3-C6 alcoholsfrom dilute aqueous solutions, such as fermentation broths. Fermentationand recovery may be conducted simultaneously. The combination ofrecovery during fermentation has several key economic advantages. Thiscombination provides improved volumetric productivity for thefermentation and allows recovery of the alcohol. An additional economicadvantage is the reduction in energy required to dry the spentfermentation broth. This reduction occurs because the batchconcentration of the alcohol product recovered for a given fermentationis raised by the simultaneous fermentation and recovery process whichincreases the quantity of alcohol produced and recovered per quantity offermentation broth dried. The term “batch concentration” refers to theconcentration for a given fermentor volume based on all of the C3-C6alcohol produced during the batch fermentation even if some of the C3-C6alcohol is removed during the fermentation. Thus, the present inventionallows for production and recovery of C3-C6 alcohols at low capital andreduced operating costs.

The term “fermentation” or “fermentation process” is defined as aprocess in which a biocatalyst is cultivated in a culture mediumcontaining raw materials, such as feedstock and nutrients, wherein thebiocatalyst converts raw materials, such as a feedstock, into products.A biocatalyst, and related fermentation processes, suitable for thepresent invention are discussed in detail in the U.S. patent applicationSer Nos. 12/263,436 and 12/263,442, filed Oct. 31, 2008, ProvisionalApplication 61/110,543, filed Oct. 31, 2008, and Provisional Application61/121,830, filed Dec. 11, 2008, which are incorporated by reference intheir entirety. The biocatalyst may be any microorganism capable ofconverting a selected feedstock to a desired C3-C6 alcohol. Furtheraspects of the biocatalyst are discussed below. Any feedstock thatcontains a fermentable carbon source is suitable for the presentinvention.

The terms fermentation broth and fermentation medium are synonymous.Unless explicitly noted, the term fermentation broth should be construedto include both fermentation broth containing micro-organisms as well asfermentation broth which does not contain microorganisms.

In one embodiment, the present invention includes a method to recover aC3-C6 alcohol from a dilute aqueous solution of the C3-C6 alcohol, suchas a fermentation broth comprising microorganisms and the C3-C6 alcohol.This method includes increasing the activity of the C3-C6 alcohol in aportion of the aqueous solution to at least that of saturation of theC3-C6 alcohol in the portion. The term saturation of the C3-C6 alcoholin the aqueous solution refers to the maximum concentration of the C3-C6alcohol under the conditions (e.g. temperature and pressure) of thataqueous solution. The method also includes forming a C3-C6 alcohol-richliquid phase and a water-rich liquid phase from the portion of theaqueous solution, and the method includes separating the C3-C6alcohol-rich phase from the water-rich phase.

In an alternative embodiment, the present invention includes a method torecover a C3-C6 alcohol from a dilute aqueous solution of the C3-C6alcohol, such as a fermentation broth comprising microorganisms and theC3-C6 alcohol. This method includes decreasing the activity of water ina portion of the aqueous solution to at least that of saturation of theC3-C6 alcohol in the portion. The method also includes forming a C3-C6alcohol-rich liquid phase and a water-rich liquid phase from the portionof the aqueous solution, and the method includes separating the C3-C6alcohol-rich phase from the water rich phase. An example of increasingthe activity of an alcohol is when an alcohol is removed selectivelycompared with water to form another phase, such as by distillation,extraction and adsorption where the other phase is gaseous, solventphase and solid adsorbent phase, respectively. Upon condensation of thegaseous phase, separation from the solvent or separation from theadsorbent, a second liquid phase is formed in which the activity of thealcohol is higher than starting solution. An example of decreasing wateractivity is when water is removed selectively compared with alcohol toform another phase, such as selective adsorption, extraction and evenfreezing of water. The result is decreasing the activity of water in thestarting solution. Some processes both increase the activity of analcohol and decrease the activity of water. For example, if ahydrophilic solute is added to an aqueous solution of an alcohol, itleads to both decreasing water activity and increasing the alcoholactivity.

As used herein the term C3-C6 alcohols refers to an alcohol containingthree, four, five or six carbon atoms, including all of the isomersthereof, and mixtures of any of the foregoing. Thus, the C3-C6 alcoholcan be selected from propanols, butanols, pentanols, and hexanols. Moreparticularly, the C3 alcohol may be 1-propanol, or 2-propanol; the C4alcohol may be 1-butanol, 2-butanol, tert-butanol (2-methyl-2-propanol),or iso-butanol (2-methyl-1-propanol); the C5 alcohol may be 1-pentanol,2-pentanol, 3-pentanol, 2-methyl-1-butanol, 3-methyl-1-butanol,2-methyl-2-butanol, 3-methyl-2-butanol, or 2,2-dimethyl-1-propanol; andthe C6 alcohol may be 1-hexanol, 2-hexanol, 3-hexanol,2-methyl-1-pentanol, 3-methyl-1-pentanol, 4-methyl-1-pentanol,2-methyl-2-pentanol, 3 methyl-2-pentanol, 4-methyl-2-pentanol,2-methyl-3-pentanol, 3-methyl-3-pentanol, 3,3-dimethyl-1-butanol,2,2-dimethyl-1-butanol, 2,3-dimethyl-1-butanol, 2,3-dimethyl-2-butanol,3,3-dimethyl-2-butanol, or 2-ethyl-1-butanol. In a preferred embodiment,the C3-C6 alcohol is iso-butanol (2-methyl-1-propanol).

A “dilute” aqueous solution as used herein means a solution containingthe C3-C6 alcohol at a concentration below the solubility limit of theC3-C6 alcohol in the solution. Concentration can be expressed in avariety of different units, e.g. weight or volume percent, molarconcentration, molal concentration or alcohol/water w/w of v/v ratio.Unless specified otherwise, however, the concentrations are presentedhere as weight percent. In case of a stream comprising at least oneadditional compound (e.g. solute, solvent, adsorbent, etc.), alcoholweight concentration as used herein is calculated by 100 times alcoholweight in that stream divided by the combined weights of alcohol andwater in that stream. In some embodiments, the ratio of the C3-C6alcohol to water in the dilute aqueous solution is less than about 10/90(w/w). In some preferred embodiments, the ratio is less than about 9/91(w/w), less than about 8/92 (w/w), less than about 7/93 (w/w), less thanabout 6/94 (w/w), less than about 5/95 (w/w), less than about 4/96(w/w), less than about 3/94 (w/w), less than about 2/98 (w/w), less thanabout 1/99 (w/w), or less than about 0.5/99.5 (w/w).

In some embodiments, the dilute aqueous solution may comprise afermentation broth. In other embodiments it may be a recycled stream ofthe fermentation broth and/or a product of the processing thereof, suchas the water rich phase from the step of separating the C3-C6alcohol-rich phase from the water-rich phase or the high boiling pointproduct comprising the C3-C6 alcohol described below or combinations ofthose with a fermentation broth. The fermentation broth may comprise theC3-C6 alcohols, along with any impurities. The term “impurity” or“impurities” means any compound other than water and the alcohol beingpurified. The term impurity includes any byproduct or co-product of thefermentation process i.e. a product related to the production ofalcohol, other than the alcohol, in any amount or in an undesiredamount. Reference herein to purification means increasing the ratiobetween a product and another compound other than water.

The method includes increasing the activity of the C3-C6 alcohol in aportion of the aqueous solution to at least that of saturation of theC3-C6 alcohol in the portion. As used herein, reference to a “portion”of a thing, such as a fermentation broth, includes both the entire thing(e.g., an entire fermentation broth) or some part of the entire thingthat is less than the entire thing (e.g., a sidestream of a fermentationbroth). A portion of a solution or fermentation broth also includes thesolution or fermentation broth if it is converted to vapor phase. Theactivity of a C3-C6 alcohol refers to the effective concentration of theC3-C6 alcohol in an aqueous solution. The activity of the C3-C6 alcoholwill depend on temperature, pressure, and composition. The activity of aspecies can be changed or modified because molecules in a non-idealsolution, such as a fermentation medium interact with each other andinteract differently with different types of molecules. For example, ifa hydrophilic solute is introduced into an aqueous isobutanol solution,the hydrophilic solute will interact with greater affinity with thewater in the solution than with the isobutanol. The activity of theisobutanol in the solution will thereby be increased. The activitycoefficient for a compound in an aqueous solution is an indicator ofwhat concentration of that compound will be in a vapor phase inequilibrium with the solution and is a function of the concentration ofthe compound in water. The activity of a compound in a solution is theproduct of the concentration of the compound and its activitycoefficient. For example, in an isobutanol-water mixture, the activitycoefficient for isobutanol is higher than water. Therefore, theconcentration of isobutanol in the vapor phase in equilibrium with theaqueous solution will be higher than in the solution.

Increasing the activity of the C3-C6 alcohol to at least that ofsaturation of the C3-C6 alcohol in an aqueous solution refers toprocessing a portion of the aqueous solution to form a compositioncomprising C3-C6 alcohol in which the effective concentration of theC3-C6 alcohol with respect to the aqueous solution is greater than inthe starting portion. This step promotes the condition that some of theC3-C6 alcohol is no longer soluble in the aqueous solution and enablesthe formation of a C3-C6 alcohol-rich liquid phase and a water-richliquid phase. Such processing can encompass a variety of process stepsincluding, but not limited to addition of a hydrophilic solute,distilling a vapor phase comprising water and the C3-C6 alcohol, reverseosmosis, dialysis, selective adsorption and solvent extraction.

As used herein, the term dialysis means selective transfer of water fromthe solution through a membrane into another compartment. Dialysisdiffers from reverse osmosis in that in reverse osmosis, water transferis induced by applying pressure on alcohol solution, while in dialysis,water transfer is induced into a compartment by having a concentratedsolution in the compartment.

According to an embodiment of the invention, increasing the activity ofthe C3-C6 alcohol may comprise adding a hydrophilic solute to theaqueous solution. In some embodiments in which the aqueous solution is afermentation broth, the hydrophilic solute may be added to the entirefermentation broth in the fermentor or to a partial stream taken fromthe fermentor, either with microorganisms in the broth or after removalof them. Reference to adding a hydrophilic solute can refer toincreasing the concentration of a hydrophilic solute already existing inthe portion of the solution or to addition of a hydrophilic solute thatwas not previously in the solution. Such increase in concentration maybe done by external addition. Alternatively, or additionally, increasingconcentration may also be conducted by in situ treatment of thesolution, such as by hydrolyzing a solute already existing in thesolution, e.g. hydrolyzing proteins to add amino acids to the solution,hydrolyzing starch or cellulose to add glucose to the solution and/orhydrolyzing hemicellulose to add pentoses to the solution. According toanother preferred embodiment, the hydrophilic solute may be one that hasa nutritional value and optionally ends up in a fermentation coproductstream, such as distillers dried grains and solubles (DDGS). In additionor alternatively, the hydrophilic solute can be fermentable and can betransferred with the water-rich liquid phase to the fermentor.

Sufficient hydrophilic solute is added to enable the formation of asecond liquid phase, either solely by addition of the hydrophilic soluteor in combination with other process steps. The required amount dependson the chemical nature of the alcohol, typically decreasing withincreasing number of carbon atoms in the alcohol and being smaller fornormal alcohols and linear ones compared with secondary or tertiaryalcohols and branched ones. The required amount further decreases withincreasing concentration of the alcohol in the fermentation liquid andpossibly also with increasing concentration of other solutes there. Theamount required in each case can be determined, in view of the presentinvention, experimentally.

Preferred hydrophilic solutes are those that have a strong effect oflowering the water partial vapor pressure of aqueous solutions. Theadded hydrophilic solute may be a salt, an amino acid, a water-solublesolvent, a sugar or combinations of those. In related embodiments, thehydrophilic solute can be recovered. For example, if the dilute aqueoussolution is fermentation broth and the hydrophilic solute added toincrease the activity of the C3-C6 alcohol in the fermentation broth isCaCl₂, then CaCl₂, after formation of alcohol-rich and water-rich liquidphases, will be primarily found the water-rich liquid phase and can berecovered therefrom.

In some embodiments, the hydrophilic solute may be a water solublecarbon source. In the embodiments in which the aqueous solution is afermentation broth, the water soluble carbon source may be added to theentire fermentation broth in the fermentor or to a partial stream takenfrom the fermentor, either with microorganisms in the broth or afterremoval of them. Reference to adding a water soluble carbon source canrefer to increasing the concentration of a water soluble carbon sourcealready existing in the portion of the solution or to addition of ahydrophilic solute that was not previously in the solution. Suchincrease in concentration may be done by external addition.Alternatively, or additionally, increasing concentration may also beconducted by in situ treatment of the solution, such as by hydrolyzing acarbon source already existing in the solution, e.g. hydrolyzingproteins to add amino acids to the solution, hydrolyzing starch orcellulose to add glucose to the solution and/or hydrolyzinghemicellulose to add pentoses to the solution. According to anotherpreferred embodiment, the water soluble carbon source may have anutritional value and optionally may end up in a fermentation coproductstream, such as distillers dried grains and solubles (DDGS).

Preferred water soluble carbon source are those that have a strongeffect of lowering the water partial vapor pressure of aqueous solutionsand ones that are well fermented. The added water soluble carbon sourcemay be a carbohydrate such as a monosaccharide, a disaccharide or anoligosaccharide and their combinations. Such saccharide may comprisehexoses, e.g. glucose and fructose and pentoses (e.g. xylose orarabinose) and their combination. Also suitable is a precursor of suchcarbohydrate, such as starch, cellulose, hemicellulose and sucrose orcombinations of those. In related embodiments, the water soluble carbonsource can be recovered or used. For example, if the dilute aqueoussolution is a portion of a fermentation broth and the water solublecarbon source added to increase the activity of the C3-C6 alcohol in thefermentation broth is glucose, then glucose will be primarily found in awater-rich liquid phase and can be conducted back to the fermentationbroth to provide carbon for fermentation.

In some embodiments, the step of increasing the activity of the C3-C6alcohol comprises distilling a vapor phase comprising water and theC3-C6 alcohol. The aqueous solution, such as a fermentation broth, canbe distilled wherein the alcohol and water are vaporized to form analcohol-depleted liquid phase and an alcohol-enriched vapor phase. Thestep of distillation can be accomplished by increasing the temperatureof the aqueous solution, reducing the atmospheric pressure on theaqueous solution or some combination thereof. The C3-C6 alcoholconcentration in the vapor phase is greater than in the aqueoussolution. According to a preferred embodiment, C3-C6 alcoholconcentration in the vapor phase is at least about 5 times greater thanthe concentration in the aqueous solution, preferably about 10 times,preferably about 15 times, preferably about 20 times, preferably about25 times, and preferably about 30 times. The vapor phase may becondensed, such as at conditions selected so that immisciblealcohol-rich and water-rich (i.e., alcohol-poor) solutions are formed.

The step of distilling can be conducted at below atmospheric pressure,at about atmospheric pressure or above atmospheric pressure. Referenceherein to atmospheric pressure is to atmospheric pressure at sea leveland unless otherwise specified, all pressures expressed herein areabsolute pressures. Suitable below atmospheric pressures includepressures from about 0.025 bar to about 1.01 bar, from about 0.075 barto about 1.01 bar, and from about 15 bar to about 1.01 bar. Suitableabove atmospheric pressures include pressures from about 1.01 bar toabout 10 bar, from about 1.01 bar to about 6 bar, and from about 1.01bar to about 3 bar.

In the embodiment when the step of distilling is conducted at belowatmospheric pressures, the temperature can be between about 20° C. andabout 95° C., between about 25° C. and about 95° C., between about 30°C. and about 95° C., or between about 35° C. and about 95° C.

In a further embodiment, in which the aqueous solution is a portion of afermentation broth and comprises microorganisms, and in which the stepof distilling is conducted in a distillation vessel, the portion of thefermentation broth is at the temperature of between about 20° C. andabout 95° C., between about 25° C. and about 95° C., between about 30°C. and about 95° C., or between about 35° C. and about 95° C. prior tointroduction into the distillation vessel. In another embodiment, thetemperature of the portion of the fermentation broth is brought to thedesired value after it is introduced in the distillation vessel.Preferably, microorganisms are used that are viable, and even morepreferably, both viable and productive at these temperatures.

Optionally, after the step of distilling, the alcohol-depleted remainingportion of the fermentation broth can be conducted from the distillationvessel to a fermentation vessel. Optionally, the alcohol-depletedremaining portion of the fermentation broth can be mixed with water,with feedstock and/or possibly other nutrients to form the culturemedium for further fermentation.

In a further embodiment, in which the portion of the aqueous solution isa portion of a fermentation broth, the step of distilling can beconducted in a fermentation vessel.

In the case where the step of increasing the activity of the C3-C6alcohol comprises distilling a vapor phase comprising water and theC3-C6 alcohol and condensing the vapor phase, the method can alsoinclude treating the portion of the dilute aqueous solution fordecreasing water activity. In various embodiments, said decreasing wateractivity comprises water removal before the step of distilling orsimultaneously with the step of distilling. The step of treating caninclude selective removal of water, selective binding of water orselective rejection of water. According to various embodiments, the stepof treating can include addition of a hydrophilic solute, addition of acarbon source, reverse osmosis, dialysis, adsorption of the alcohol on aselective adsorbent, extraction of the alcohol into a selectiveextractant, adsorption of water on a selective adsorbent, or extractionof water into a selective extractant.

In a further embodiment in which the step of increasing the activity ofthe C3-C6 alcohol comprises distilling a vapor phase comprising waterand the C3-C6 alcohol and condensing the vapor phase, the method canalso include treating C3-C6 alcohol-rich phase for water removal. Thestep of treating can include selective removal of water, selectivebinding of water or selective rejection of water. Alternatively, thestep of treating can include addition of a hydrophilic solute, additionof a carbon source, reverse osmosis, dialysis, adsorption of the butanolon a selective adsorbent, extraction of the butanol into a selectiveextractant, adsorption of water on a selective adsorbent, or extractionof water into a selective extractant.

In a preferred embodiment, the step of distilling is conducted in aflash tank, that can be operatively connected to a fermentation vesseland the process can further comprise circulating the culture medium fromthe fermentation vessel to the flash tank, and circulating the culturemedium from the flash tank to the fermentation vessel. A flash is a onestage distillation where the vapor and liquid outlet from the flashsystem are in equilibrium with each other and the temperature andpressure of each phase is nearly identical. Distillation, on the otherhand, comprises a series of flash stages strung together sequentially.During distillation i.e. in a multi stage flash system, such as adistillation column, the vapor that comes out the top and the liquidthat comes out the bottom leave at different temperatures than in aflash.

According to another embodiment, the process includes reducing pressurein a distillation vessel compared with that in the fermentation vessel.Such a pressure reduction coupled with adiabatic vaporization allows forremoval of heat from the portion of the fermentation broth of theaqueous solution generated in the fermentation vessel within thedistillation vessel. Alternatively or in addition, the process caninclude increasing pressure on the aqueous solution from thedistillation vessel in the fermentation vessel. Such a pressure increasecreates heat, which can be used to preheat the system at various points.For example, the heat can be used to preheat the feed in the flash tank,the beer still and/or the distillation column and can also be used inthe evaporators used to concentrate the thin stillage to syrup. Thesecomponents are discussed in detail below.

Flash tank vacuum evaporation operations have less engineering concernsregarding pressure drop under vacuum because the flash tank acts as asingle stage of separation without stages of liquid above the flash tankimpacting pressure drop on the system, and the differential pressureacross flash tank operations can be very low. Design calculations forvapor generation in the flash tank and sizing of piping systems can beappropriately selected to achieve low pressure drop. The distillation ofa C3-C6 alcohol in a flash tank requires less vacuum than a distillationcolumn and, thus, the flash tank has lower operating cost and capitalcosts inasmuch as the equipment is smaller in size and simpler inconstruction.

In a preferred embodiment, when the step of increasing the activity ofthe C3-C6 alcohol comprises distilling a vapor phase comprising waterand the C3-C6 alcohol, the mixed vapor includes an azeotropiccomposition. Azeotropes are formed when molecular forces cause two ormore molecular species to behave as a new vapor or/liquid species.Azeotropes are generally viewed as a limitation by chemical processindustries because the azeotrope composition “pinch point” prevents thedistillation of the mixture into pure components. Instead of producingpure components from the distillation process, the azeotrope manifestsitself as an azeotropic composition at the top of the distillationcolumn, as a minimum boiling point azeotrope, or from the bottom of thedistillation column, as a maximum boiling point azeotrope.

When fermentation products form a maximum boiling point azeotrope withwater, all of the non-azeotrope bound water must be vaporized anddistilled overhead. Products within fermentation broth are typicallydilute. As a result, when maximum boiling point azeotropes are formed,the amount of energy required to boil up and remove the excess un-boundwater is a large heat load and can often make the vaporization andcondensation processes of distillation uneconomical. Additionally, themaximum boiling point azeotrope occurs at temperatures above the boilingpoints of the pure species, elevating the bottom temperatures in thedistillation system. As a result, the bottoms product in the maximumboiling point experiences a higher heat history than the pure species.This high temperature heat history can degrade the value of the primaryproduct and co-products of fermentation. Distiller's dry grains andsolubles (DDGS), which are typically used as a feed ingredient, are oneexample of such a co-product which can be degraded with exposure to highheat and lose nutritional values.

Minimum boiling point azeotropes are also known as positive azeotropesbecause the azeotrope has an activity coefficient of greater than 1.Maximum boiling point azeotropes are also referred to as negativeazeotropes because their activity coefficient is less than 1. Themagnitude of the activity coefficient dictates the degree of non-idealactivity of the azeotropic entity. This non-ideality and difficulty inseparation of azeotropes has been studied. The activity coefficient isnot fixed but is a function of concentration of the compound in water.As a result, the solution boiling point of the azeotrope compositionvaries as the concentration of the component varies. As a result, theazeotropic composition developed in the vapor phase can be affected bycomponent concentration and operating pressure.

According to a preferred embodiment, an aqueous solution of the C3-C6alcohol forms a minimum boiling point azeotrope. According to a relatedpreferred embodiment, the concentration of the C3-C6 alcohol in themixed vapor is substantially equal to the concentration of the alcoholin the minimum boiling point azeotrope at the pressure selected fordistillation. In some particularly preferred embodiments, theconcentration of the C3-C6 alcohol in the mixed vapor is greater thanthe concentration of the alcohol in the minimum boiling point azeotrope,as in some cases where the aqueous solution comprises other solutes inaddition to the alcohol that affect the water partial vapor pressure.

Some azeotropes are known to be stable under a broad range of operatingpressures, while other azeotrope systems can be “broken” by low and highpressure. For example, the ethanol-water azeotrope is broken atpressures less than 70 torr. For azeotropes that can be broken undervacuum, the use of distillation columns is sometimes limited due to thefact that the vacuum distillation columns require that the pressure dropin the distillation column is significant enough that it requires deepervacuum to be pulled at the vacuum source. For example, attempting tomaintain the vacuum distillation column feed pressure to 150 mm Hgrequires that the pressure drop in the column be very small so as toensure that the vacuum pump can maintain proper vacuum levels. Toachieve low pressure drop in vacuum columns with multiple trays requiressmall liquid heights on the distillation trays. The low pressure dropand low liquid height in the column typically increases the columncapital cost by increasing the diameter of the column.

In some embodiments, the step of increasing the activity of the C3-C6alcohol comprises dialysis. Dialysis works on the principle of diffusionof solutes and ultra-filtration of fluid across a semi-permeablemembrane. Any membrane separation system that selectively removes waterfrom the aqueous solution is suitable for the process of the presentinvention. According to a preferred embodiment, dialysis is conducted ina system comprising two or more compartments. The aqueous solution ofthe alcohol is introduced into one and water from this solutiontransfers selectively through the membrane into the other. According toa preferred embodiment, the water transfer is induced by osmoticpressure. The water-receiving compartment contains a hydrophiliccompound, e.g. CaCl₂ or a carbohydrate, or a concentrated solution ofsuch compound. A concentrated solution is formed in the water-receivingcompartment. That solution is treated according to various embodimentsto regenerate the solute or its concentrated solution, or for otherapplications. Regeneration can be done by known means such as waterdistillation. In the case where the solute is a carbohydrate or anothersource of fermentable carbon, the solution can be used providefermentables to the fermentation step.

In some embodiments, the step of increasing the activity of the C3-C6alcohol comprises reverse osmosis. In reverse osmosis, the aqueoussolution is contacted in a first compartment with a reverse osmosismembrane under pressure, whereby water selectively transfers through themembrane to a second compartment, while the alcohol is retained in thefirst compartment. As a result of selective water transfer into thesecond compartment, the concentration (and activity) of the alcohol inthe liquid of the first compartment increases and preferably reachessaturation, whereby a second phase is formed in that first compartment.That compartment comprises according to this embodiment two liquidphases one of which is an alcohol-saturated aqueous phase and the otheris a water saturated alcohol solution.

In some embodiments, the step of increasing the activity of the C3-C6alcohol comprises solvent extraction. In solvent extraction, the aqueoussolution is contacted with another liquid phase (solvent or extractant),wherein at least one of water and the alcohol are not fully miscible.The two phases are mixed and then allowed to settle. According to oneembodiment, the step of increasing the activity of the C3-C6 alcoholcomprises extraction of the C3-C6 alcohol into an alcohol-selectiveextractant. The term “alcohol-selective extractant” means an extractantpreferring alcohol over water so that the alcohol/water ratio in theextractant is greater than in the remaining aqueous solution. Thus, thealcohol-selective extractant or solvent is selective to the alcohol(similarly or more hydrophobic than the alcohol) and the alcoholtransfers preferentially into the extractant or solvent to formalcohol-containing extractant or solvent, also referred to as extract.In some preferred embodiments, the alcohol-selective solvent may bebutylacetate, tributylphosphate, decanol, 2-heptanone or octane. Inanother embodiment, the step of increasing the activity of the C3-C6alcohol comprises extraction of water into a water selective extractant.The term “water-selective extractant” means an extractant preferringwater over alcohol so that the alcohol/water ratio in the extractant islower than in the remaining aqueous solution. Thus, the water-selectiveextractant or solvent is selective to water (more hydrophilic than thealcohol), so that water transfers preferentially into thewater-selective extractant or solvent.

In a preferred embodiment, the alcohol-selective solvent can be anacidic, amine based extractant. Such an extractant can be prepared bymixing an amine with a diluent and contacting the mixture with an acid.Amines that are suitable for forming the extractant include primary,secondary, tertiary and quaternary amines, and preferably includeprimary, secondary, tertiary amines. Suitable amines are alsowater-insoluble in both free and salt form (i.e. when an acid is boundto them). Preferably the aggregate/total number of carbon atoms on theamines is at least 20. Both aliphatic and aromatic amines are suitableand aliphatic ones are preferred. The diluent can be a hydrocarbon oranother non-reactive organic solvent with boiling point of at leastabout 60° C., and preferably at least about 80° C. The acid can be anystrong acid, such as one with a pKa (−log dissociation constant) of notgreater than 3, and can either be a mineral acid or an organic acid. Inone example, the amine can be trioctyl amine, the acid can be sulfuricacid and the diluent can be decane. The acid is extracted (binds to theamine) to form the extractant.

In some embodiments the step of increasing the activity of the C3-C6alcohol comprises adsorption of the C3-C6 alcohol or water on aselective adsorbent. In adsorption, the aqueous solution is contactedwith a selective adsorbent that has greater selectivity for eitheralcohol or water. In one embodiment, the step of increasing the activityof the C3-C6 alcohol comprises adsorption of the C3-C6 alcohol on analcohol selective adsorbent. An “alcohol-selective adsorbent” means anadsorbent preferring alcohol over water so that the alcohol/water ratioon the adsorbent is greater than in the remaining aqueous solution. Inanother embodiment, the step of increasing the activity of the C3-C6alcohol comprises adsorption of water on a water-selective adsorbent. A“water-selective adsorbent” means an adsorbent preferring water overalcohol so that the alcohol/water ratio on the adsorbent is lower thanin the remaining aqueous solution. Thus, the aqueous phase is contactedwith a water-selective adsorbent, a water-carrying adsorbent is formedand the aqueous solution is enriched in the C3-C6 alcohol. According tovarious embodiments, the water adsorbent is hydrophilic, has surfacefunctions capable of forming hydrogen bonds and/or has pores suitable insize to the size of water molecules. In some embodiments the adsorbentmay be solid. According to a preferred embodiment, a fermentationfeedstock, such as ground corn may be the adsorbent. For example, thefeedstock may be contacted with the aqueous solution to selectivelyadsorb water out of it. In some embodiments the adsorbent may be amolecular sieve.

The method further includes the step of forming a C3-C6 alcohol-richliquid phase and a water-rich liquid phase from the portion of theaqueous solution which has been treated to increase the activity of theC3-C6 alcohol. As used here, the term “alcohol-rich liquid phase” meansa liquid phase wherein the alcohol-to-water ratio is greater than thatin the portion of the aqueous solution. The term “water-rich liquidphase” means a liquid phase wherein the water-to-alcohol ratio isgreater than that of the alcohol-rich liquid phase. The water-rich phaseis also referred to in the following as alcohol-lean phase. The step offorming the two phases can be active. For example, in some embodiments,the step of forming may comprise condensing a distilled vapor phase thatforms two phases after condensation. Alternatively or in addition,chilling or cooling the treated portion of the aqueous solution canresult in the formation of the two phases. Other steps for activelyforming the two phases can include using equipment shaped to promote theseparation of phases. Separation of the phases can be accomplished invarious unit operations including liquid-liquid separators comprising aliquid/liquid separator utilizing specific gravity differences betweenthe phases and a water boot, g-force separation as in a centrifuge, orcentrifugal liquid-liquid separators. Also suitable are settlers as inmixer settler units used for solvent extraction processes. In someembodiments the step of forming is passive and may simply be a naturalconsequence of the previous step of increasing the activity of the C3-C6alcohol to at least that of saturation.

In the alcohol-rich liquid phase, the ratio of the concentration of theC3-C6 alcohol with respect to the water is effectively greater than inthe starting portion. In the water-rich phase, the ratio ofconcentration of the C3-C6 alcohol with respect to water is effectivelyless than in the alcohol-rich liquid phase. The water-rich phase mayalso be referred to as the alcohol-poor phase.

In some embodiments, the C3-C6 alcohol is propanol and the weight ratioof propanol to water in the alcohol-rich phase is greater than about0.2, greater than about 0.5, or greater than about 1. In someembodiments, the C3-C6 alcohol is butanol and the ratio of butanol towater in the alcohol-rich phase is greater than about 1, greater thanabout 2, or greater than about 8. In some embodiments, the C3-C6 alcoholis pentanol and the ratio of pentanol to water in the alcohol-rich phaseis greater than about 4, greater than about 6, or greater than about 10.

The concentration factor or enrichment factor for a given phase can beexpressed as the ratio of alcohol to water in that phase divided by theratio of alcohol to water in the dilute aqueous solution. Thus, forexample, the concentration or enrichment factor for the alcohol-richphase may be expressed as the ratio of alcohol/water in the alcohol-richphase divided by that ratio in the aqueous dilute solution.

In some embodiments, the ratio of the C3-C6 alcohol to water in theC3-C6 alcohol-rich phase is greater than the ratio of the C3-C6 alcoholto water in the fermentation broth by at least about 5 fold, at leastabout 25 fold, at least about 50 fold, at least about 100 fold, or atleast about 300 fold.

The process further includes separating the C3-C6 alcohol-rich phasefrom the water-rich phase. Separating the two phases refers to physicalseparation of the two phases and can include removing, skimming, pouringout, decanting or otherwise transferring one phase from another and maybe accomplished by any means known in the art for separation of liquidphases.

In some embodiments, the method further comprises the step of coolingthe C3-C6 alcohol-rich phase to increase the ratio of the C3-C6 alcoholto water in the alcohol-rich phase.

In some embodiments, the method further comprises recovering the C3-C6alcohol from the alcohol-rich phase. Recovering refers to isolating theC3-C6 alcohol from the alcohol-rich phase. Recovering also includesenriching or increasing the concentration of the C3-C6 alcohol in thealcohol-rich phase. In various embodiments, this step may comprise aprocess selected from the group consisting of distillation, dialysis,water adsorption (e.g., such as use of molecular sieves), solventextraction, contact with a hydrocarbon liquid that is immiscible inwater and contact with a hydrophilic compound to produce a first phasecomprising the C3-C6 alcohol and water and a second phase comprisingC3-C6 alcohol, wherein the ratio of water to C3-C6 alcohol in the secondphase is less than in the first phase. In preferred embodiments, thesecond phase comprises at least about 80%, about 85%, about 90%, about95% or about 99% by weight C3-C6 alcohol. As used herein a liquid thatis immiscible in water has a miscibility in water of less than about 1wt %.

Methods of distillation and dialysis are discussed above with respect tothe step of increasing the activity of C3-C6 alcohols and similarprocesses can be used to recover C3-C6 alcohol from a C3-C6 alcohol-richphase. Regarding the use of water adsorption to recover C3-C6 alcoholfrom a C3-C6 alcohol-rich phase, the alcohol-rich phase is contactedwith an adsorbent that selectively adsorbs water out of the alcohol richphase. A water-carrying adsorbent is formed and the alcohol-rich phaseis further enriched in the C3-C6 alcohol. According to variousembodiments, the water adsorbent is hydrophilic, has surface functionscapable of forming hydrogen bonds and/or has pores suitable in size tothe size of water molecules. In some embodiments the adsorbent may besolid. According to a preferred embodiment, a fermentation feedstock,such as ground corn may be the adsorbent. For example, the feedstock maybe contacted with the C3-C6 alcohol-rich phase to selectively adsorbwater out of it. In some embodiments the adsorbent may be a molecularsieve.

Solvent extraction can also be used to recover C3-C6 alcohol from aC3-C6 alcohol-rich phase. In solvent extraction, the alcohol-rich phaseis contacted with another liquid phase (solvent), wherein at least oneof water and the alcohol are not fully miscible. The two phases aremixed and then allowed to settle. According to one embodiment, thesolvent is selective to water (more hydrophilic than the alcohol), watertransfers preferentially to the solvent phase and the alcohol-to-waterratio in the other phase increases. According to another embodiment, thesolvent is selective to the alcohol (similarly or more hydrophobic thanthe alcohol). In some preferred embodiments the alcohol-selectivesolvent may be butylacetate, tributylphosphate, decanol, 2-heptanone oroctane. The alcohol transfers preferentially into the solvent. In afollowing step, the alcohol is separated from the solvent in a formhaving higher alcohol-to-water ratio compared with that of thealcohol-rich phase.

Contact with a hydrocarbon liquid that is immiscible in water can alsobe used to recover C3-C6 alcohol from a C3-C6 alcohol-rich phase. Suchliquids are hydrophobic solvents and act as described above forhydrophobic solvents, i.e. extracting the alcohol from the alcohol-richphase. Examples of such hydrocarbon liquids include gasoline, crude oil,Fischer Tropsch materials and biofuels.

Contact with a hydrophilic compound can also be used to recover C3-C6alcohol from a C3-C6 alcohol-rich phase. This method for recovery issimilar to that described above for use of a hydrophilic compound toincrease alcohol activity or to decrease water activity.

In the case of distillation to recover C3-C6 alcohol from a C3-C6alcohol-rich phase, the first phase comprises a vapor phase comprisingC3-C6 alcohol and water and the second phase comprises a high boilingproduct comprising C3-C6 alcohol.

In a further embodiment of the present invention, the process caninclude after the step of increasing the activity, conducting (ortransporting) the remaining portion of the dilute aqueous solution, suchas a fermentation broth, to a fermentation vessel. In this embodiment,the remaining portion of the dilute aqueous solution can comprise animpurity and the process further includes removing at least a portion ofthe impurity from at least a portion of the remaining portion beforeconducting the remaining portion to the fermentation vessel. Suchimpurities can be, for example, ethanol, acetate, aldehydes such asbutyraldehyde, and short chain fatty acids. In some embodiments, thedilute aqueous solution can include an impurity and the ratio of theimpurity to the C3-C6 alcohol in the C3-C6 alcohol-rich liquid phase isgreater than the ratio in the water-rich phase. In some embodiments, theratio of the impurity to the C3-C6 alcohol in the C3-C6 water-richliquid phase is greater than the ratio in the alcohol-rich phase.

In further embodiments of the invention, the C3-C6 alcohol-rich phase isfurther processed to increase the value or utility of the phase. Otherembodiments of further processing are disclosed in U.S. patentapplication Ser. No. 12/327,723, filed Dec. 3, 2008, which isincorporated by reference in its entirety. For example, the C3-C6alcohol-rich phase can be further processed by (i) distillingsubstantially pure C3-C6 alcohol from the C3-C6 alcohol-rich phase, (ii)distilling an azeotrope of the C3-C6 alcohol from the C3-C6 alcohol-richphase, (iii) contacting the C3-C6 alcohol-rich phase with a C3-C6alcohol-selective adsorbent; or (iv) combining the C3-C6 alcohol-richphase with a hydrocarbon liquid that is immiscible in water. In the caseof distilling substantially pure C3-C6 alcohol from the C3-C6alcohol-rich phase, the substantially pure C3-C6 alcohol can have a lowproportion of impurities (such as reflected by having a low ratio ofimpurities to C3-C6 alcohol). For example, the ratio of impurities toC3-C6 alcohol, in the substantially pure C3-C6 alcohol can be less thanabout 5/95, less than about 2/98, or less than about 1/99. Alternativelythe substantially pure C3-C6 alcohol can have a water content of lessthan about 5 wt %, less than about 1 wt % or less than about 0.5 wt %.

In the case of combining the C3-C6 alcohol-rich phase with a hydrocarbonliquid that is immiscible in water, the resulting combination can form asingle uniform phase. Alternatively, in the case of combining the C3-C6alcohol-rich phase with a hydrocarbon liquid that is immiscible inwater, the combination can form a light phase and a heavy phase and theratio of C3-C6 alcohol to water in the light phase is greater than inthe heavy phase. According to an embodiment of the method, thehydrocarbon liquid is a fuel, such as gasoline. According to a relatedembodiment, a C3-C6 alcohol-enriched fuel is formed by combining a fuelwith a C3-C6 alcohol-rich phase, further comprising water. As a resultof combining the C3-C6 alcohol selectively transfers into the fuel phaseto form said enriched fuel, whereas the majority of the water containedinitially in the alcohol-rich phase separates as a water-rich heavyphase, which is separated from the fuel.

In other embodiments of the invention, in which the dilute aqueoussolution is a fermentation medium, the process can include a step ofculturing a microorganism in a fermentation medium to produce the C3-C6alcohol before the steps of increasing the activity of the C3-C6 alcoholin a portion of the fermentation medium, forming a C3-C6 alcohol-richliquid phase and a water-rich phase and separating the C3-C6alcohol-rich phase from the water-rich phase. In one preferredembodiment, the invention further includes the step of conducting thewater rich phase to the fermentation medium before the step ofincreasing the activity of the alcohol. In such embodiments, the step ofculturing can be a process selected from a batch fermentation, afed-batch fermentation, a continuous fermentation, a cell recyclefermentation, and an enzyme reaction process.

As noted above, a suitable biocatalyst, and related fermentationprocesses, are discussed in detail in the U.S. patent application Ser.Nos. 12/263,436 and 12/263,442, filed Oct. 31, 2008, provisionalapplication 61/110,543, filed Oct. 31, 2008 and provisional application61/121,830, filed Dec. 11, 2008. Suitable microorganisms can be selectedfrom naturally occurring microorganisms, genetically engineeredmicroorganisms and microorganisms developed by classical techniques, ora combination thereof. Such microorganisms can include, withoutlimitation, bacteria and fungi (including yeast). For example, suitablebacteria can include those that are capable of alcohol production suchas the bacteria of the Clostridium species. Examples of these includewithout limitation, Clostridium butyricum, Clostridium acetobutylicum,Clostridium saccharoperbutylacetonicum, Clostridium saccharobutylicumand Clostridium beijerinckii.

Suitable bacteria and fungi also include those that are capable ofhydrolyzing carbohydrates and can be genetically engineered to producealcohols. Examples include, without limitation, bacteria of the orderClostridiales (e.g. Butyrovibrio fibrisolvens), Bacilliales (e.g.Bacillus circulars), Actinomycetales (e.g. Streptomyces cellulolyticus),Fibrobacterales (e.g. Fibrobacter succinogenes), Xanthomonadales(Xanthomonas species) and Pseudomonadales (e.g. Pseudomonas mendocina)and fungi such as those of the order Rhizopus, Saccharomycopsis,Aspergillus, Schwanniomyces and Polysporus. The fungi may be able to dothe conversion aerobically or anaerobically. Examples of anaerobic fungiinclude, without limitation, Piromyces species (e.g. strain E2),Orpinomyces species (e.g. Orpinomyces bovis), Neocallimastix species (N.frontalis), Caecomyce species, Anaeromyces species and Ruminomycesspecies.

Microorganisms provided herein are modified to produce metabolites inquantities not available in the parental microorganism. A “metabolite”refers to any substance produced by metabolism or a substance necessaryfor or taking part in a particular metabolic process. A metabolite canbe an organic compound that is a starting material (e.g., glucose orpyruvate), an intermediate (e.g., 2-ketoisovalerate), or an end product(e.g., isobutanol) of metabolism. Metabolites can be used to constructmore complex molecules, or they can be broken down into simpler ones.Intermediate metabolites may be synthesized from other metabolites,perhaps used to make more complex substances, or broken down intosimpler compounds, often with the release of chemical energy.

Exemplary metabolites include glucose, pyruvate, and isobutanol. Themetabolite isobutanol can be produced by a recombinant microorganismmetabolically engineered to express or over-express a metabolic pathwaythat converts pyruvate to isobutanol. An exemplary metabolic pathwaythat converts pyruvate to isobutanol may be comprised of an acetohydroxyacid synthase (ALS) enzyme encoded by, for example, alsS from B.subtilis, a ketolacid reductoisomerase (KARI) encoded by, for exampleilvC from E. coli, a dihydroxy-acid dehydratase (DHAD), encoded by, forexample ilvD from E. coli, a 2-keto-acid decarboxylase (KIVD) encodedby, for example kivd from L. lactis, and an isobutyraldehydedehydrogenase (IDH), encoded by, for example, by a native E. colialcohol dehydrogenase.

Accordingly, provided herein are recombinant microorganisms that produceisobutanol and in some aspects may include the elevated expression oftarget enzymes such as ALS (encoded e.g. by the ilvIH operon from E.coli), KARI (encoded e.g. by ilvC from E. coli), DHAD (encoded, e.g. byilvD from E. coli), and KIVD (encoded, e.g. by PDC6 from S. cerevisiae,ARO10 from S. cerevisiae, THI3 from S. cerevisiae, kivd from L. lactis,or pdc from Z. mobilis).

The microorganism may further include the deletion or inhibition ofexpression of enzymes such as an ethanol dehydrogenase (encoded, e.g.,by an adhE gene), Idh (encoded, e.g., by an IdhA), frd (encoded, e.g.,by an frdB, an frdC or an frdBC gene), fnr, leuA, ilvE, poxB, ilvA,pflB, or pta gene, or any combination thereof, to increase theavailability of pyruvate or reduce enzymes that compete for a metabolitein a desired biosynthetic pathway.

In yeast microorganisms, pyruvate decarboxylase (PDC) is a majorcompetitor for pyruvate. During anaerobic fermentation, the main pathwayto oxidize the NADH from glycolysis is through the production ofethanol. Ethanol is produced by alcohol dehydrogenase (ADH) via thereduction of acetaldehyde, which is generated from pyruvate by pyruvatedecarboxylase (PDC). Thus, most of the pyruvate produced by glycolysisis consumed by PDC and is not available for the isobutanol pathway.Another pathway for NADH oxidation is through the production ofglycerol. Dihydroxyacetone-phosphate, an intermediate of glycolysis, isreduced to glycerol 3-phosphate by glycerol 3-phosphate dehydrogenase(GPD). Glycerol 3-phosphatase (GPP) converts glycerol 3-phosphate toglycerol. This pathway consumes carbon from glucose as well as reducingequivalents (NADH) resulting in less pyruvate and reducing equivalentsavailable for the isobutanol pathway. These pathways contribute to lowyield and low productivity of isobutanol. Accordingly, deletions of PDCand GPD may increase yield and productivity of isobutanol.

The disclosure identifies specific genes useful in the methods,compositions and organisms of the disclosure; however it will berecognized that absolute identity to such genes is not necessary. Forexample, changes in a particular gene or polynucleotide comprising asequence encoding a polypeptide or enzyme can be performed and screenedfor activity. Typically such changes comprise conservative mutation andsilent mutations. Such modified or mutated polynucleotides andpolypeptides can be screened for expression of a functional enzyme usingmethods known in the art.

Due to the inherent degeneracy of the genetic code, otherpolynucleotides which encode substantially the same or a functionallyequivalent polypeptide can also be used to clone and express thepolynucleotides encoding such enzymes.

As will be understood by those of skill in the art, it can beadvantageous to modify a coding sequence to enhance its expression in aparticular host. The genetic code is redundant with 64 possible codons,but most organisms typically use a subset of these codons. The codonsthat are utilized most often in a species are called optimal codons, andthose not utilized very often are classified as rare or low-usagecodons. Codons can be substituted to reflect the preferred codon usageof the host, a process sometimes called “codon optimization” or“controlling for species codon bias.”

Optimized coding sequences containing codons preferred by a particularprokaryotic or eukaryotic host (see also, Murray et al. (1989) Nucl.Acids Res. 17:477-508) can be prepared, for example, to increase therate of translation or to produce recombinant RNA transcripts havingdesirable properties, such as a longer half-life, as compared withtranscripts produced from a non-optimized sequence. Translation stopcodons can also be modified to reflect host preference. For example,typical stop codons for S. cerevisiae and mammals are UAA and UGA,respectively. The typical stop codon for monocotyledonous plants is UGA,whereas insects and E. coli commonly use UAA as the stop codon (Dalphinet al. (1996) Nucl. Acids Res. 24: 216-218). Methodology for optimizinga nucleotide sequence for expression in a plant is provided, forexample, in U.S. Pat. No. 6,015,891, and the references cited therein.

Those of skill in the art will recognize that, due to the degeneratenature of the genetic code, a variety of DNA compounds differing intheir nucleotide sequences can be used to encode a given enzyme of thedisclosure. The native DNA sequence encoding the biosynthetic enzymesdescribed above are referenced herein merely to illustrate an embodimentof the disclosure, and the disclosure includes DNA compounds of anysequence that encode the amino acid sequences of the polypeptides andproteins of the enzymes utilized in the methods of the disclosure. Insimilar fashion, a polypeptide can typically tolerate one or more aminoacid substitutions, deletions, and insertions in its amino acid sequencewithout loss or significant loss of a desired activity. The disclosureincludes such polypeptides with different amino acid sequences than thespecific proteins described herein so long as they modified or variantpolypeptides have the enzymatic anabolic or catabolic activity of thereference polypeptide. Furthermore, the amino acid sequences encoded bythe DNA sequences shown herein merely illustrate embodiments of thedisclosure.

In addition, homologues of enzymes useful for generating metabolites areencompassed by the microorganisms and methods provided herein.

As used herein, two proteins (or a region of the proteins) aresubstantially homologous when the amino acid sequences have at leastabout 30%, 40%, 50% 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity. To determine the percentidentity of two amino acid sequences, or of two nucleic acid sequences,the sequences are aligned for optimal comparison purposes (e.g., gapscan be introduced in one or both of a first and a second amino acid ornucleic acid sequence for optimal alignment and non-homologous sequencescan be disregarded for comparison purposes). In one embodiment, thelength of a reference sequence aligned for comparison purposes is atleast 30%, typically at least 40%, more typically at least 50%, evenmore typically at least 60%, and even more typically at least 70%, 80%,90%, 100% of the length of the reference sequence. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The disclosure provides metabolically engineered microorganismscomprising a biochemical pathway for the production of isobutanol from asuitable substrate at a high yield A metabolically engineeredmicroorganism of the disclosure comprises one or more recombinantpolynucleotides within the genome of the organism or external to thegenome within the organism The microorganism can comprise a reduction,disruption or knockout of a gene found in the wild-type organism and/orintroduction of a heterologous polynucleotide and/or expression oroverexpression of an endogenous polynucleotide.

In one aspect, the disclosure provides a recombinant microorganismcomprising elevated expression of at least one target enzyme as comparedto a parental microorganism or encodes an enzyme not found in theparental organism. In another or further aspect, the microorganismcomprises a reduction, disruption or knockout of at least one geneencoding an enzyme that competes with a metabolite necessary for theproduction of isobutanol. The recombinant microorganism produces atleast one metabolite involved in a biosynthetic pathway for theproduction of isobutanol. In general, the recombinant microorganismscomprises at least one recombinant metabolic pathway that comprises atarget enzyme and may further include a reduction in activity orexpression of an enzyme in a competitive biosynthetic pathway. Thepathway acts to modify a substrate or metabolic intermediate in theproduction of isobutanol. The target enzyme is encoded by, and expressedfrom, a polynucleotide derived from a suitable biological source. Insome embodiments, the polynucleotide comprises a gene derived from abacterial or yeast source and recombinantly engineered into themicroorganism of the disclosure.

It is understood that a range of microorganisms can be modified toinclude a recombinant metabolic pathway suitable for the production ofisobutanol. In various embodiments, microorganisms may be selected frombacterial or yeast microorganisms. Microorganisms for the production ofisobutanol at high yield may be selected based on certaincharacteristics:

One characteristic may include the ability to metabolize a carbon sourcein the presence of isobutanol. For example, a microorganism may have ahigher yield that metabolizes a carbon source at 30 g-isobutanol L⁻¹ ata rate equal to or higher than a microorganism that metabolizes a carbonsource at 20 g-isobutanol L⁻¹. Similarly, a microorganism thatmetabolizes a carbon source at a rate equal to or higher than 40g-isobutanol L⁻¹ may have a higher yield than a microorganism thatmetabolizes a carbon source at 30 g-isobutanol L⁻¹. Additionally, amicroorganism that metabolizes a carbon source at a rate equal to orhigher than 50 g-isobutanol L⁻¹ may have a higher yield than amicroorganism that metabolizes a carbon source at 40 g-isobutanol L⁻¹.Generally, a higher yield is more desirable than a lower yield.

Another characteristic may include the property that the microorganismis selected to convert various carbon sources into isobutanol.Accordingly, in one embodiment, the recombinant microorganism hereindisclosed can convert a variety of carbon sources to products, includingbut not limited to glucose, galactose, mannose, xylose, arabinose,lactose, sucrose, and mixtures thereof.

Another characteristic specific to a yeast microorganism may include theproperty that the microorganism is able to metabolize a carbon source inthe absence of pyruvate decarboxylase (PDC). In an embodiment, it ispreferable that the yeast microorganism is able to metabolize 5- and6-carbon sugar in the absence of PDC. In one embodiment, it is even morepreferred that a yeast microorganism is able to grow on 5- and 6-carbonsugars in the absence of PDC.

Yeast microorganisms may be selected from a number of genera, includingbut not limited to Saccharomyces spp., including S. cerevisiae and S.uvarum, Kluyveromyces spp., including K. thermotolerans, K. lactis, andK. marxianus, Pichia spp., Hansenula spp., including H. polymorpha,Candida spp., Trichosporon spp., Yamadazyma spp., including Y. stipitis,Torulaspora spp., including T. pretoriensis, Schizosaccharomyce spp.,including S. pombe, Cryptococcus spp., Aspergillus spp., Neurospora spp.or Ustilago spp., Dekkera spp., Brettanomyces spp. In exemplaryembodiments, genera are Kluyveromyces and Saccharomyces whereas examplemicroorganisms are Kluyveromyces lactis and Saccharomyces cerevisiae.

Bacterial Microorganisms may be selected from a number of genera,including but not limited to Arthrobacter, Bacillus, Brevibacterium,Clostridium, Corynebacterium, Escherichia, Gluconobacter, Lactobacillus,Nocardia, Pseudomonas, Rhodococcus, Saccharomyces, Shewanella,Streptomyces, Xanthomonas, and Zymomonas. In another embodiment, suchhosts are E. coli or Pseudomonas. In another embodiment, such hosts areE. coli W3110, E. coli B, Pseudomonas oleovorans, Pseudomonasfluorescens, or Pseudomonas putida.

One exemplary metabolic pathway for the conversion of glucose toisobutanol begins with the conversion of glucose to pyruvate viaglycolysis. Glycolysis also produces 2 moles of NADH and 2 moles of ATP.Two moles of pyruvate are then used to produce one mole of isobutanol.Alternative isobutanol pathways have been described in InternationalPatent Application No PCT/US2006/041602 and in Dickinson et al., Journalof Biological Chemistry 273:25751-15756 (1998).

Accordingly, the engineered isobutanol pathway to convert pyruvate toisobutanol can be, but is not limited to, the following reactions:

1. 2 pyruvate→+acetolactate+CO₂

2. acetolactate+NADPH→+2,3-dihydroxyisovalerate+NADP⁺

3. 2,3-di hydroxyisovalerate→+alpha-ketoisovalerate

4. alpha-ketoisovalerate→+isobutyraldehyde+CO₂

5. isobutyraldehyde+NADPH→+isobutanol+NADP⁺

These reactions are carried out by the enzymes 1) Acetolactate Synthase(ALS), 2) Ketol-acid Reducto-Isomerase (KARI), 3) Dihydroxy-aciddehydratase (DHAD), 4) Keto-isovalerate decarboxylase (KIVD), and 5) anIsobutyraldehyde Dehydrogenase (IDH).

In one aspect, the two enzymes within an isobutanol biosynthetic pathwaythat convert pyruvate to isobutanol that utilize a NADPH cofactor may bereplaced with ones that utilize NADH. These two enzymes may be KARI andIDH or an alcohol dehydrogenase. The NADPH-dependent KARI and IDHenzymes may be engineered using directed evolution and site-directedmutagenesis.

In another embodiment, the microorganism is engineered to overexpressthese enzymes. For example, ALS can be encoded by the alsS gene of B.subtilis, alsS of L. lactis, or the ilvK gene of K. pneumonia. Forexample, KARI can be encoded by the ilvC genes of E. coli, C.glutamicum, M. maripaludis, or Piromyces sp E2. For example, DHAD can beencoded by the ilvD genes of E. coli or C. glutamicum. KIVD can beencoded by the kivd gene of L. lactis. ADH can be encoded by ADH2, ADH6,or ADH7 of S. cerevisiae.

The microorganism of the invention may be engineered to have increasedability to convert pyruvate to isobutanol. In one embodiment, themicroorganism may be engineered to have increased ability to convertpyruvate to isobutyraldehyde. In another embodiment, the microorganismmay be engineered to have increased ability to convert pyruvate toketo-isovalerate. In another embodiment, the microorganism may beengineered to have increased ability to convert pyruvate to2,3-dihydroxyisovalerate. In another embodiment, the microorganism maybe engineered to have increased ability to convert pyruvate toacetolactate.

As noted above, any microorganism, whether naturally occurring ormanmade, that is capable of producing alcohol can be used and themethods of the present invention are not limited to the examples listedhere. In some embodiments, the microorganism is viable at temperaturesfrom about 20° C. to about 95° C. Reference to a microorganism beingviable at a given temperature or range of temperatures refers to amicroorganism being able to survive exposure to such temperatures andsubsequently be able to grow and/or produce metabolic products under thesame or different conditions. In other embodiments, the microorganism isa temperature resistant microorganism. The term “resistance” is definedas the property of a biocatalyst to have a low rate of inhibition in thepresence of increasing concentrations of an inhibitor in thefermentation broth. The term “more resistant” describes a biocatalystthat has a lower rate of inhibition towards an inhibitor than anotherbiocatalyst with a higher rate of inhibition towards the same inhibitor.For example, two biocatalysts A and B, both with a tolerance of 2% to aninhibitor biofuel precursor and a specific productivity of 1 g productper g CDW per h, exhibit at 3% biofuel precursor a specific productivityof 0.5 g product per g CDW per h and 0.75 g product per g CDW per h forA and B, respectively. The biocatalyst B is more resistant than A. Theterm “temperature resistant” describes a biocatalyst that has a lowerrate of inhibition at a given temperature than another biocatalyst witha higher rate of inhibition at the same temperature.

The term “tolerance” is defined as the ability of the biocatalyst tomaintain its specific productivity at a given concentration of aninhibitor. The term “tolerant” describes a biocatalyst that maintainsits specific productivity at a given concentration of an inhibitor. Forexample, if in the presence of 2% of an inhibitor a biocatalystmaintains the specific productivity that it had at 0 to 2%, thebiocatalyst is tolerant to 2% of the inhibitor or has a tolerance to 2%of the inhibitor. The term “tolerance to temperature” is defined as theability of the biocatalyst to maintain its specific productivity at agiven temperature.

In some embodiments, the microorganism has a productivity of at leastabout 0.5 g/L per hour of the C3-C6 alcohol in aggregate over thelifetime of a batch fermentation cycle. In some embodiments, theproductivity is at least about 1, at least about 1.5, at least about2.0, at least about 2.5, at least about 3.0, at least about 3.5, atleast about 4.0, at least about 4.5, and at least about 5.0 g/L per hourof the C3-C6 alcohol in aggregate over the lifetime of a batchfermentation cycle. In some embodiments, the productivity ranges fromabout 0.5 g/L per hour to about 5 g/L per hour of the C3-C6 alcohol overthe lifetime of a batch fermentation cycle.

In other embodiments, preferred microorganisms are ones that produce thedesired alcohol with no or minimal coproducts or byproducts. Alsopreferred are microorganisms that use simple and low cost fermentationmedia.

Any feedstock that contains a fermentable carbon source is suitable forembodiments of the present invention that include a step of culturing amicroorganism. Examples include feedstocks containing polysaccharides,such as starch, cellulose and hemicellulose, feedstocks containingdisaccharides, such as sucrose, sugarcane juice and sucrose-containingmolasses, and monosaccharides, such as glucose and fructose. Suitablefeedstocks include starchy crops, such as corn and wheat, sugarcane andsugar beet, molasses and lignocellulosic material. Suitable feedstocksalso include algae and microalgae. Where desired, the feedstock mayundergo treatments such as comminution, milling, separation of thecarbon source from other components, such as proteins,decrystallization, gelatinization, liquefaction, saccharification, andhydrolysis catalyzed by means of chemical and/or enzymatic catalysts.Such treatment can be conducted prior to fermenting or simultaneouslywith it, e.g. as in simultaneous saccharification and fermentation.

The fermentation broth of the present invention typically has a singleliquid phase, but is not necessarily homogeneous since it may containnon-fermented insoluble solids, e.g. in a suspended form. Thefermentation feedstock may contain compounds of limited water solubilityand optionally also of limited or no fermentability. For example,according to an embodiment of the invention, the fermentation feedstockis comminuted corn and the carbon source is starch contained in it.Possibly, the starch is gelatinized, liquefied and/or saccharified, butinsoluble components whether starchy or others (e.g. non-fermentedprotein) may still exist in the fermentation liquid. According toanother embodiment, the fermentation feedstock is a lignocellulosicmaterial and the carbon source is hydrolyzed cellulose and/orhemicellulose. Here again, some of the feedstock components are oflimited water solubility. In these and other cases, the fermentationliquid may consist of an aqueous solution of the alcohol with solidssuspended in it. Yet, according to an important aspect of the invention,in all those cases, only a single liquid phase exists in thefermentation broth.

A further embodiment of the invention is a method to produce a C3-C6alcohol that includes hydrolyzing a feedstock that comprises apolysaccharide and at least one other compound to produce fermentablehydrolysis products, a portion of which are fermented in a fermentationmedium to produce the C3-C6 alcohol. In this embodiment, thefermentation medium further comprises at least one non-fermentedcompound. The method further comprises increasing the activity of theC3-C6 alcohol from a portion of the fermentation medium, forming a C3-C6alcohol-rich liquid phase and a water-rich phase, and separating theC3-C6 alcohol-rich phase from the water-rich phase, as described abovein earlier embodiments of the invention. This method further includesseparating the at least one non-fermented compound from the fermentationmedium, the water-rich phase or both. For example, the at least onenon-fermented compound can include a material such as DDGS.

In various embodiments of the invention that include fermentation, thestep of fermentation can be conducted simultaneously with other processsteps such as various recovery methods disclosed herein, that includethe steps of increasing the activity of a C3-C6 alcohol and also thesteps of hydrolyzing feed stocks to prepare a fermentation substrate.

In this method, the step of hydrolyzing can include any method capableof breaking polymeric carbohydrates into fermentable products. Thus, thestep of hydrolyzing may be chemically or enzymatically catalyzedhydrolysis or autohydrolysis, and saccharification. In this method, thesteps of hydrolyzing and fermenting can be conducted simultaneously forat least a portion of time of the method, can be conductedsimultaneously for all the time of the method, or can be conducted atdistinct times.

In a particular embodiment of this method, the step of fermenting isconducted with a microorganism that is capable of hydrolyzing thefeedstock. Suitable microorganisms can be selected from naturallyoccurring microorganisms, genetically engineered microorganisms andmicroorganisms developed by classical techniques, or a combinationthereof. and have been discussed in detail above.

An alternative embodiment of the present invention is a method toproduce a C3-C6 alcohol that includes culturing a microorganism in afermentation medium to produce the C3-C6 alcohol. The step of culturingis described in detail above. The method further includes increasing theactivity of the C3-C6 alcohol in a portion of the fermentation mediumand distilling the portion of the fermentation medium to produce a vaporphase comprising water and C3-C6 alcohol and a liquid phase. The stepsof increasing the activity and distilling are discussed above in regardto other embodiments of the present invention. The method furtherincludes conducting the liquid phase resulting from the distillationstep (the depleted liquid phase) to the fermentation medium. In apreferred embodiment, the portion of the fermentation medium in whichthe activity of the C3-C6 alcohol is increased comprises microorganismsthat remain in the depleted liquid phase and are returned to thefermentation medium for further production of C3-C6 alcohol by themicroorganism. In some embodiments, the liquid phase comprises animpurity and the method further includes removing at least a portion ofthe impurity from at least a portion of the liquid phase before the stepof conducting the liquid phase to the fermentation medium. Inembodiments of this method, the ratio of the C3-C6 alcohol to water inthe portion of the fermentation medium is less than about 10/90 (w/w),less than about 7.5/92.5 (w/w), less than about 5.0/95 (w/w), less thanabout 2.5/97.5 (w/w), less than about 2/98 (w/w), less than about1.5/98.5 (w/w), less than about 1/99 (w/w), or less than about 0.5/99.5(w/w).

A further alternative embodiment of the present invention is a method torecover a C3-C6 alcohol from a dilute aqueous solution comprising thesteps of distilling a portion of the dilute aqueous solution to a vaporphase comprising C3-C6 alcohol and water and condensing the vapor phase.

In this embodiment, the vapor phase comprises between about 1% by weightand about 45% by weight of the C3-C6 alcohol that is present in theportion of the dilute aqueous solution. While it is possible to distillmore than 45% of the C3-C6 alcohol from the portion of the diluteaqueous solution into the vapor phase, by controlling or limiting theamount of alcohol in the solution that is distilled to the vapor phase(i.e., leaving a significant amount of alcohol behind), a number ofimportant advantages are achieved. As a greater portion of the alcoholin the solution is distilled, the amount of water relative to the amountof alcohol is increased resulting in the need to handle increased waterload downstream which can result in increased energy requirements. Inaddition, for example, in the context of a fermentation, thenon-distilled portion of the solution can be returned to thefermentation vessel and used as part of the fermentation medium forproduction of additional alcohol that can be recovered in a similarprocess. This process, therefore, is highly efficient because it allowsfor alcohol recovery in the step of distillation in a range in which therelative amount of alcohol to water is high. In various alternativeembodiments, the vapor phase can comprise between about 2% by weight andabout 40% by weight of the C3-C6 alcohol, between about 3% by weight andabout 35% by weight of the C3-C6 alcohol and between about 4% by weightand about 30% by weight of the C3 C6 alcohol and between about 5% byweight and about 25% by weight of the C3-C6 alcohol present in theportion of the dilute aqueous solution.

In some embodiments, the step of distilling comprises a single stagedistillation. Single stage distillation may take place in a flash tank.The design of a flash tank has been described in detail above.

In some embodiments, the ratio of the C3-C6 alcohol to water in thedilute aqueous solution is less than about 10/90 (w/w). In somepreferred embodiments, the ratio is less than about 7.5/92.5 (w/w), lessthan about 5.0/95 (w/w), less than about 2.5/97.5 (w/w), less than about2/98 (w/w), less than about 1.5/98.5 (w/w), less than about 1/99 (w/w),or less than about 0.5/99.5 (w/w).

The step of distilling may be adiabatic or isothermal. In adiabaticdistilling no significant heat transfer takes place between thedistillation system and the surroundings, and the pressure of the systemis held constant. In isothermal distilling heat transfer is allowedbetween the distillation system and the surroundings, and thetemperature of the system is held constant.

In various embodiments of this method, the enrichment of alcohol fromthe dilute aqueous solution to the vapor is at least about 5 fold, about6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, about11 fold, about 12 fold, about 13 fold, about 14 fold or about 15 fold.The term “enrichment” refers to the ratio of alcohol/water in thecondensed vapor divided by the ratio of alcohol/water in the aqueousdilute solution.

In some embodiments, the method further comprises the step of forming aC3-C6 alcohol-rich liquid phase and a water-rich liquid phase from theportion of the dilute aqueous solution. In some embodiments, the methodfurther comprises separating the C3 C6 alcohol-rich phase and thewater-rich phase. The steps of forming an alcohol-rich liquid phase anda water-rich liquid phase, and of separating the two have been discussedin detail above.

A further alternative embodiment of the present invention is a method toproduce a product from a C3-C6 alcohol in a dilute aqueous solution,such as a fermentation broth comprising microorganisms and the C3-C6alcohol. This method includes distilling a vapor phase comprising waterand C3-C6 alcohol from the dilute aqueous solution and reacting theC3-C6 alcohol in the vapor phase to form the product. This embodimentfurther comprises other process steps disclosed herein leading to theformation of the vapor phase.

An embodiment of the present invention is shown in FIG. 1. Fermentationis conducted in fermentor 10. The fermentation broth in the fermentor 10includes the C3-C6 alcohol product, such as butanol, and othercomponents of the fermentation medium. During the course of thefermentation, a stream of the fermentation broth, which may includemicroorganisms, is conducted from the fermentor 10 to a heat exchanger20 via 12. The heat exchanger 20 is used to raise the temperature of thefermentation broth to a temperature suitable for a subsequentdistillation. After the temperature of the fermentation broth is raisedto an appropriate temperature, the broth is further conducted to a flashtank 30 for distillation via 22. The fermentation heat can partiallysupply the heat required for vaporization in the flash system. The flashtank 30 is maintained at a below atmospheric pressure so that uponintroduction of the heated fermentation broth into the flash tank 30, aportion of the fermentation broth gets vaporized. The portion of thevaporized fermentation broth includes only a portion of the butanol inthe fermentation broth along with water vapor. After distillation in theflash tank 30, the remaining portion of the fermentation broth that isnot distilled is returned to the fermentor 10 via 34. This fermentationbroth that is being returned to the fermentor is now partially depletedof butanol. The portion of the fermentation broth that is vaporized inthe flash tank 30 is conducted as a vapor to a vapor condenser 40 via32, which can be cooled, for example, by chilled water via 42. Uponcondensation of the mixed butanol and water vapor, the condensedsolution is conducted to a phase separator 50 via 44. The remainingvapor that is not condensed is then further conducted to an outlet via48. The condensed solution in the phase separator is allowed to separateinto a heavy liquid phase and a light liquid phase. The heavy liquidphase consists primarily of water with some amount of butanol soluble inthe water. The light phase consists primarily of butanol with someamount of soluble water. From the phase separator, the light phasecontaining butanol can be recovered by separation from the heavy phaseand can be treated for further purification. The heavy phase consistingprimarily of water can be conducted for other applications or uses inthe system. 13, 35 are liquid pumps and 47 is a vacuum pump.

With reference to FIG. 2, a specific embodiment of the present inventionis presented, illustrating the production of butanol by simultaneoussaccharification and fermentation of pretreated corn, and azeotropicdistillation of a side stream of butanol. Dry corn is milled into a finepowder. The milled (ground) corn 1, thin stillage 3, CIP fermentorcleanout 31, recycled water 43, and steam 2 are added to a corn starchpretreatment system 32 where the mixture is slurried and heated to about99° C. (A CIP (Clean in Place) fermentor cleanout is a caustic watersolution that is used to clean and sanitize the fermentors betweenbatches. NaOH is often used but other strong bases and othersanitization chemicals can also be used. The waste CIP solution containssolids, nutrients, carbohydrates etc from the fermentor (clinging towalls) that can be reintroduced into the front end of the cornpretreatment.) Alpha-amylase 50 is added to the corn starch pretreatmentsystem 32 where the holding time can be about 1 hour or less.Glucoamylase enzyme 4 is added after the solution is cooled to atemperature ranging from about 50° C. to about 65° C. After a shortsaccharification time of about 5-6 hours the slurry is cooled to about32° C. The slurry solids concentration at this point can be about 361g/kg, including insoluble and soluble solids. Enzymes 4 sufficient tocomplete the saccharification in about 32 hours are also added to thecorn mash mixture, which is transferred to the fermentor 5. Thefermentation is run under simultaneous saccharification and fermentation(SSF) mode at 32° C. A side stream 6 containing about 4 wt. % butanol iscontinuously removed from the fermentor 5 and a flash tank heatexchanger 33 is used to control the temperature of a flash tank feed 7at about 34° C. Vacuum of about 50 mm Hg is pulled on a flash tank 34and an azeotropic vapor composition 11 is formed. The composition of thebutanol water vapor azeotrope 11 can be about 54 wt % butanol and about46 wt % water. The azeotrope vapor 11 is pumped by the vacuum pump 35and is either fed to a chemical conversion process 13 or to a condenser12. The condensed vapor phase 36 is conducted to a liquid/liquidseparator 37 where it is phase separated. The condensed vapor phaseseparates into a butanol rich phase 37 a and a water rich phase 37 b.The butanol rich phase 37 a has a butanol concentration of about 680 g/Lbutanol. The water rich phase 37 b has a butanol concentration of about86 g/L. The ratio of the volumes produced for the upper layer 37 a tothe lower layer 37 b is 3 to 1.

The unvaporized components 9 in the flash tank 34 including cells,water, nutrients, carbohydrates, and about 2 wt % unvaporized butanolare returned to the fermentor 5. The unvaporized components 9 aredepleted of butanol and when returned to the fermentor 5, can continueto produce butanol to be recovered by treatment of the side stream 6 asdescribed above.

The water rich heavy phase 37 b from the liquid/liquid separator 37 isconducted 15 to a beer still 38 and distilled. A butanol-waterazeotropic composition 18 is generated in the beer still 38 and isconducted to a condenser 39 to be condensed. The condensed vapor 19 isconducted to a liquid/liquid separator 40 to be separated into a waterrich heavy phase 40 b and a butanol rich light phase 40 a. The waterrich heavy phase 40 b contains about 86 g/L butanol is recycled 20 backto the beer still 38. The butanol rich phase 40 a has a butanolconcentration of about 680 g/L butanol.

The butanol rich light phase 40 a in the liquid/liquid separator 40 isconducted 21 to a distillation system 41. The butanol rich light phase37 a in the liquid/liquid separator 37 is also conducted 16 to thedistillation system 41, and can be combined with the butanol rich lightphase 40 a. The distillation system 41 is operated at atmosphericpressure and purified butanol is produced as a high boiling product 22at a concentration of about 99 wt % butanol. (In other embodiments, thedistillation system can be operated at sub atmospheric, atmospheric, orsuper atmospheric pressures.) A butanol water azeotrope vapor 23 isproduced and sent to the condenser 45 and condensed. The condensed vapor46 is conducted to a liquid/liquid separator 47 to be separated into awater rich heavy phase 47 b and a butanol rich light phase 47 a. Thewater rich heavy phase 47 b is recycled 48 to the beer still 38. Thebutanol rich light phase 47 a is conducted 51 to the distillation system41 and can be combined with other inputs 16, 21.

The SSF fermentation in the fermentor 5 is conducted for 52 hours. Thefermentation broth containing about 2% butanol that is not removed bythe vacuum flash tank 34 is conducted 8 to the beer still 38. Thebutanol in the broth is distilled overhead as a butanol-water azeotrope18. From the beer still 38, water, unconverted carbohydrates, nutrients,cells, fiber, corn germ, enzymes, and other fermentation components aretaken as a bottoms product 17 and contains about 0.05 wt % butanol. Thebeer still bottoms stream 17 is divided to a distillers dry grain dryer27 and a purge stream 28. Thin stillage 3 is produced by the purgestream 28. Dried distillers grains 29 are produced by the dryer 27. Thedryer 27 also produces water vapor 30 that is condensed by a condenser42 and recycled 43 to the corn starch pretreatment system 32.

The fermentor 5, condenser 12 (having an inflow from the flash tank 34),condenser 39 (having an inflow from the beer still 38), and condenser 45(having an inflow from the distillation system 41) have vent streams 10,25, 24, 49 that contain butanol, water, CO₂, and other inert gases.These streams are combined in a vent collection system 44 and areprocessed in downstream equipment 26 to recover and purify butanol andCO₂.

The foregoing embodiment of the invention can be conducted in a retrofitcorn ethanol production plant in which the primary operations, includingcorn starch pretreatment system, fermentor, beer still, distillationsystem, and dryer are operations that previously were used to produceethanol. Such systems have multiple fermentors (typically from five toseven) that are operated in cycle so that each one conducts afermentation for about 52 hours before being emptied into a beer still.The operations upstream of the fermentors (e.g., the corn starchpretreatment system) operate essentially continuously preparing afeedstock for a first fermentor and then preparing a feedstock for asecond fermentor and so forth. The operations downstream of thefermentors (e.g., the beer still, distillation system, and dryer)operate essentially continuously taking the fermentation broth from eachfermentor as it finishes a fermentation cycle to recover ethanol,produce DDGS, a purge stream and thin stillage.

Such an ethanol production plant can be retrofitted to produce butanolby incorporating various production and recovery processes as describedherein. Typically, microorganisms that produce ethanol are tolerant tohigh concentrations of ethanol in the fermentation broth. However, highconcentrations of C3-C6 alcohols in the fermentation broth can be toxicto microorganisms. Therefore, a low cost method to simultaneously removealcohols as they are produced is required to operate an ethanol plant toproduce a C3-C6 alcohol instead of ethanol.

Since butanol concentrations cannot be generated that are as high asethanol concentrations before butanol production organisms shut down,the production and recovery processes described herein are useful forincorporation into an ethanol plant to allow efficient production ofbutanol. By incorporating butanol recovery processes in which a portionof a fermentation broth that can include microorganisms is taken to arecovery operation such as a flash tank for recovery of a portion of thebutanol from the portion of the fermentation broth and returning abutanol-depleted stream to a fermentor, the effective butanolconcentration of the fermentation can be significantly increased so thata butanol production process can be conducted into an ethanol productionplant.

The process of retrofitting a plant can include introducing equipment toproduce a side stream 6, flash tank feed 7, and unvaporized componentsstream 9, as described above into a plant. In addition, equipment forconducting liquid/liquid separations such as separators 37, 40, can beintroduced to provide for efficient recovery of butanol.

Accordingly, in one embodiment, the present invention includes a methodto operate a retrofit ethanol production plant to produce a C3-C6alcohol. In this embodiment, the retrofit ethanol production plantcomprises a pretreatment unit, multiple fermentation units, and a beerstill to produce the C3-C6 alcohol. The method includes the steps ofpretreating a feedstock to form fermentable sugars in the pretreatmentunit; fermenting the fermentable sugars with a microorganism thatproduces the C3-C6 alcohol in a fermentation medium in a firstfermentation unit; treating a portion of the fermentation medium toremove the C3-C6 alcohol; returning the treated portion to the firstfermentation unit; and transferring the fermentation medium from thefirst fermentation unit to the beer still.

The method includes the step of pretreating a feedstock to formfermentable sugars in the pretreatment unit. The pretreatment unitcontinuously receives the feedstock for pretreatment. The termpretreatment refers to treatments such as comminution, milling,separation of the carbon source from other components such as proteins,decrystallization, gelatinization, liquefaction, saccharification, andhydrolysis catalyzed by means of chemical and/or enzymatic catalysts.For example, the feedstock may be dry corn which may be ground, mixedwith water, heated and reacted with amylases in the pretreatment unit toproduce a mash or slurry containing fermentable sugars that are suitableas substrate for fermentation by microorganisms.

The method further includes the step of fermenting the fermentablesugars with a microorganism that produces the C3-C6 alcohol in afermentation medium in a first fermentation unit. A fermentation unitcontains fermentation medium comprising microorganisms that are capableof converting the fermentable sugars into the C3-C6 alcohol. Suchmicroorganisms have been described in detail above. The retrofit plantcomprises multiple fermentation units. A stream of the pretreatedfeedstock containing fermentable sugars from the pretreatment unit isintroduced into the first fermentation unit, where it is combined withthe fermentation medium comprising microorganisms. The microorganismsferment the fermentable sugars present to produce the C3-C6 alcohol.

The method further includes the step of treating a portion of thefermentation medium to remove the C3-C6 alcohol. The fermentation mediumcomprises the C3-C6 alcohol, water, as well as the microorganisms. Aportion (e.g., a side stream) of the fermentation medium from the firstfermentation unit is taken to remove the C3-C6 alcohol containedtherein. Treating can include any one or more of the methods forpurification and recovery of C3-C6 alcohols from dilute aqueoussolutions described herein and specifically, can include the steps ofdistilling a vapor phase comprising water and C3-C6 alcohol, addition ofa hydrophilic solute, addition of a water soluble carbon source, reverseosmosis, and dialysis, and mixtures thereof, all of which steps havebeen described in detail above. In a preferred embodiment, this stepcomprises directing a sidestream from the first fermentation unit to aflash tank where the step of distilling is conducted at belowatmospheric pressures. The design of a flash tank has been described indetail above.

The method further includes the step of returning the treated portion tothe first fermentation unit. The treated portion is depleted in theC3-C6 alcohol and comprises water and can include microorganisms, bothof which are returned to the fermentation medium. By removing a portionof the C3-C6 alcohol from fermentation medium and returning the mediumto the fermentor, the concentration of the C3-C6 alcohol in thefermentation broth is maintained below a concentration that isdetrimental to further production of the C3-C6 alcohol.

The method further includes the step of transferring the fermentationmedium from the fermentation unit to a beer still. This step isconducted when it is desired to have the fermentation completed.Fermentation completion occurs when all fermentable carbohydrates areconsumed or when the rate of carbohydrate conversion is reduced suchthat termination of the fermentation is desired.

In some embodiments of this method, the rate of pretreating is the sameas for the plant when it produced ethanol and/or the same as forconventional ethanol plants. As used herein, reference to a rate beingthe “same” includes the rate being identically the same, but also beingwithin (plus or minus) about 25% of the rate, within about 15% of therate, within about 10% of the rate, within about 9% of the rate, withinabout 8% of the rate, within about 7% of the rate, within about 6% ofthe rate, within about 5% of the rate, within about 4% of the rate,within about 3% of the rate, within about 2% of the rate, within about1% of the rate. Thus, if the retrofit ethanol plant had a pretreatmentrate of about 115 metric tons per hour, a pretreatment rate within about25% of that rate would include a rate from about 7.5 tons per hour toabout 12.5 tons per hour. The rate of pretreating refers to the rate atwhich pretreated feedstock is conducted to a fermentation unit.

In some other embodiments of this method, the cycle time for afermentation unit is the same as for the plant when it produced ethanoland/or the same as for conventional ethanol plants. The cycle timerefers to the time from introduction of an inoculum to the time ofemptying the fermentor to a beer still. For example, a typical cycletime for a fermentor is about 52 hours.

In one embodiment, the C3-C6 alcohol output of the retrofit plant is atleast about 80% of the C3-C6 alcohol equivalent of the ethanol maximumoutput of the plant before retrofit. In other embodiments, the C3-C6alcohol output of the retrofit plant is at least about 81%, at leastabout 82%, at least about 83%, at least about 84%, at least about 85%,at least about 86%, at least about 87%, at least about 88%, at leastabout 89%, at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99% ofthe C3-C6 alcohol equivalent of the ethanol maximum output of the plantbefore retrofit.

The maximum output of an alcohol plant is a measure of the amount ofalcohol produced by that plant, and may be expressed as gallons ofalcohol produced per year or other units measuring volume or weight pertime period. The output of a plant depends on the size and design of thespecific plant. The term “ethanol maximum output of the plant beforeretrofit” refers to the maximum amount of ethanol produced by a plant orfor which the plant was engineered before it is retrofit to produce aC3-C6 alcohol.

As recognized above, microorganisms used for production of ethanol aretolerant to high concentrations of ethanol in the fermentation broth,but microorganisms used for production of C3-C6 alcohols are typicallynot tolerant to high concentrations of C3-C6 alcohols. Advantageously,using the methods of the present invention it is possible to retrofit anethanol plant to produce a C3-C6 alcohol at output levels comparable tothat of ethanol, limited only by the theoretical conversion efficiencyof that particular alcohol. The theoretical conversion efficiency ofglucose to ethanol, on a weight basis, is 51% or 0.51. (In practicehowever, some of the glucose is used by the micro-organisms forproduction of cell mass and metabolic products other than the alcohol,and the actual conversion efficiency is less than the theoreticalmaximum.) Depending on the fermentation pathway used by themicro-organism, the theoretical conversion efficiency of glucose topropanol can range from 0.33 to 0.44, that of butanol can range from0.27 to 0.41, that of pentanol can range from 0.33 to 0.39, and that ofhexanol can range from 0.28 to 0.38. The term “C3-C6 alcohol equivalent”refers to the ratio of the theoretical conversion efficiency of aparticular C3-C6 alcohol to that of ethanol and is specific for thefermentation pathway used. Thus, the “iso-butanol equivalent of ethanol”(for the pathway in which one molecule of glucose is broken into onemolecule of isobutanol, two molecules of ATP and two molecules of CO₂)as used herein is 0.401÷0.51=0.806. For example, consider an ethanolplant with an ethanol maximum output of the plant before retrofit ofabout 100 million gallons/year. Using the methods of the presentinvention, it is possible to retrofit the plant and operate it toproduce butanol at a theoretical maximum output of about 80.6 milliongallons per year. However, given that the density of ethanol is 0.7894and the density of isobutanol is 0.8106, the actual theoretical maximumoutput of isobutanol is about 78 million gallons per year. The exactnumber of gallons per year can be calculated using the densityinformation, the theoretical yields and/or the actual practical yieldsachieved.

In various embodiments, an ethanol plant can be retrofit and operated atan output of at least about 80% of the theoretical maximum output forany given C3-C6 alcohol, accounting for density differences. In otherembodiments, the C3-C6 alcohol output of the retrofit plant could be atleast about 81%, at least about 82%, at least about 83%, at least about84%, at least about 85%, at least about 86%, at least about 87%, atleast about 88%, at least about 89%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99% of theoretical maximum output, accounting fordensity differences.

Another embodiment of the invention is a method for extraction of aC3-C6 alcohol from an aqueous solution that includes contacting anaqueous solution with an acidic, amine-based extractant. The acidicamine-based extractant can be formed by acidifying an organic aminesolution as described above. Upon contact of the aqueous solution withthe extractant, the extraction is carried out by mixing the acidic,amine-based extractant with the aqueous solution. The C3-C6 alcohol canbe recovered from an extractant phase that forms after contact.

Various aspects of the invention are described in detail in the examplesprovided below. However, these examples are provided for the purpose ofillustration and are not intended to limit the scope of the presentinvention. Each publication and reference cited herein is incorporatedherein by reference in its entirety. While various embodiments of thepresent invention have been described in detail, it is apparent thatmodifications and adaptations of those embodiments will occur to thoseskilled in the art. It is to be expressly understood, however, that suchmodifications and adaptations are within the scope of the presentinvention, as set forth in the following exemplary claims.

EXAMPLES Example 1 Enrichment of C3-C6 Alcohols from Aqueous SolutionsUsing Solvents

This example illustrates the enrichment of C3-C6 alcohols, such aspropanol (PrOH), butanol (BuOH), isobutanol (i-BuOH) and pentanol(PenOH), from aqueous solutions using various solvents.

Several aqueous solutions were prepared for each alcohol, differing intheir alcohol concentrations, all of which were sub-saturated. Each ofthose aqueous solutions was mixed with a solvent, such as butylacetate,tributylphosphate, decanol, 2-heptanone or octane. The phase ratiobetween the aqueous solution and the solvent differed from one case tothe other. Mixing was continued until equilibrium was reached. Atequilibrium, phase separation could be observed forming an upper,alcohol-rich phase (light phase) and a lower, alcohol-lean phase (heavyphase). Both phases were analyzed for alcohol and water content. Thealcohol concentrations were analyzed by high pressure liquidchromatography (HPLC) at 25° C. using a YMC-Pack ODS-AM column Theeluents were methanol-water solutions, in which the methanolconcentration was 20%, 30%, 30% and 40% for propanol, butanol,isobutanol and pentanol, respectively. The water content of the organicphases was determined by the Karl-Fischer method. These analyticalmethods were also used for the analyses in the other examples.

The distribution coefficient of the alcohol was calculated for eachexperiment by dividing the alcohol concentration in the light phase bythe alcohol concentration in the heavy phase. Similarly, thedistribution coefficient for water was calculated by dividing the waterconcentration in the light phase by the water concentration in the heavyphase. The enrichment factor was calculated by dividing the distributioncoefficient of alcohol by the distribution coefficient of water. Inaddition, the alcohol to water ratio (w/w) was calculated for the lightphase. The results are reported in tables 1.1 to 1.8.

TABLE 1.1 Enrichment of isobutanol (i-BuOH) by contact with butylacetate at 25° C. Distribution Light phase Heavy phase coefficientsi-BuOH/ i- Butyl i- i-BuOH H₂O H₂O BuOH H₂O acetate BuOH H₂O EnrichmentWt % Wt % w/w Wt % Wt % Wt % Da Dw factor 0.83 1.3 0.63 0.34 99.7 0.392.5 0.013 187 3.1 1.9 1.7 1.5 98.4 0.31 2.0 0.019 105 4.4 3.1 1.4 2.597.5 0.34 1.8 0.032 57

TABLE 1.2 Enrichment of iso-butanol (i-BuOH) by contact with tri-butylphosphate (TBP) at 25° C. Distribution Light phase Heavy phasecoefficients i- i-BuOH/ i- i- BuOH H₂O H₂O BuOH H₂O TBP BuOH EnrichmentWt % Wt % w/w Wt % Wt % Wt % Da H₂O Dw factor 1.2 7.1 0.17 0.22 99.80.016 5.4 0.071 76 5.6 6.5 0.86 1.2 98.8 0.023 4.9 0.066 74 9.8 6.3 1.61.9 98.1 0.024 5.2 0.064 82

TABLE 1.3 Enrichment of iso-butanol (i-BuOH) by contact with decanol at25° C. Distribution Light phase Heavy phase coefficients i- i-BuOH/ i-i- BuOH H₂O H₂O BuOH H₂O Decanol BuOH H₂O Enrichment Wt % Wt % w/w Wt %Wt % Wt % Da Dw factor 1.11 3.8 0.29 0.30 99.7 0.0020 3.8 0.038 99 7.54.4 1.7 1.4 98.6 0.0043 5.2 0.045 116 12.0 4.6 2.6 2.2 97.8 0.0053 5.50.047 119

TABLE 1.4 Enrichment of iso-butanol (i-BuOH) by contact with 2-Heptanoneat 25° C. Distribution Light phase Heavy phase coefficients i- i-BuOH/i- 2- i- BuOH H₂O H₂O BuOH H₂O Heptanone BuOH H₂O Enrichment Wt % Wt %w/w Wt % Wt % Wt % Da Dw factor 1.15 1.7 0.68 0.44 99.2 0.40 2.6 0.017155 5.4 2.5 2.2 1.7 98.0 0.34 3.3 0.026 125 8.4 3.5 2.4 2.0 97.7 0.294.1 0.036 116

TABLE 1.5 Enrichment of propanol (PrOH) by contact with octane at 24° C.PrOH in Water in PrOH in light the light heavy Water in the phase phasephase heavy phase Enrichment Wt % Wt % Wt % Wt % Da Dw factor 0.03 <0.050.66 99.3 0.047 <0.0005 >100 0.07 <0.05 1.9 98 0.037 <0.0005 >80 0.24<0.05 5.7 94.3 0.042 <0.0005 >80

TABLE 1.6 Enrichment of iso-butanol (i-BuOH) by contact with octane at24° C. Distribution Light phase Heavy phase coefficients i- i-BuOH/ i-i- Enrich- BuOH H₂O H₂O BuOH H₂O BuOH H₂O ment Wt % Wt % w/w Wt % Wt %Da Dw factor 0.10 0.048 2.1 0.64 99.4 0.15 0.0005 300 0.30 0.059 5.1 1.998.1 0.16 0.0006 250 2.5 0.076 33 4.6 95.4 0.54 0.0008 650

TABLE 1.7 Enrichment of butanol (BuOH) by contact with octane at 24° C.Light phase Distribution BuOH/ Heavy phase coefficients En- BuOH H₂O H₂OBuOH H₂O BuOH H₂O richment Wt % Wt % w/w Wt % Wt % Da Dw factor 0.110.06 1.8 0.65 99.4 0.17 0.0006 250 0.29 0.05 5.9 1.8 98.2 0.16 0.0005300 2.5 0.07 36 4.6 95.4 0.55 0.0008 650

TABLE 1.8 Enrichment of Pentanol (PenOH) by contact with octane at 24°C. Light phase Distribution PenOH/ Heavy phase coefficients PenOH H₂OH₂O PenOH H₂O PenOH H₂O Enrichment Wt % Wt % w/w Wt % Wt % Da Dw factor0.63 0.06 10.5 0.59 99.4 1.08 0.006 180 2.95 0.08 37 1.5 98.5 2.0 0.0367 12.6 0.50 25 2.7 97.3 4.6 0.13 35

The results show that the enrichment is dependent on the alcohol and onthe selected solvent. For example, for the lower molecular weightalcohols, the more polar solvents, such as butyl acetate and tri-butylphosphate had higher distribution coefficients compared with those of aless polar solvent, such as octane. On extraction by octane, alcoholswith higher molecular weights extracted better. For a given alcohol andsolvent combination, the distribution coefficients and the enrichmentfactor were dependent on the alcohol concentration in the equilibriumaqueous phase. Yet, for all tested solvents a high enrichment factor wasobserved. The light or upper phase was the alcohol-rich phase in allcases. The alcohol to water ratio was 50 to 600 times higher in thelight phase as compared to the heavy phase.

Example 2 Extraction of Isobutanol from Aqueous Solutions Using Solvents

This example illustrates the efficiency of the enrichment of isobutanolfrom sub-saturated aqueous solutions using various solvents. Table 2summarizes the results of various isobutanol enrichment experiments.Isobutanol was extracted from aqueous solutions of various startingconcentrations. Several solvents were used to carry out a single stepextraction at 25° C. In addition, the solvent phase to aqueous phaseratio was varied. The isobutanol concentration in the solvent wasmeasured for each experiment and the extraction yield was calculated foreach experiment by dividing the alcohol amount in the solvent by theinitial alcohol amount in the aqueous solution.

TABLE 2 Enrichment of i-BuOH from aqueous solutions of variousconcentrations with several extractants: butyl acetate (BuAc), decanol(DeOH), tri-butyl-phosphate (TBP) and 2-heptanone. Initial i- Final i-i-BuOH BuOH conc. Solvent/ BuOH conc. conc. (Wt %) in aqueous (Wt %) in(Wt %) in Extraction aqueous solution aqueous solvent yield solutionSolvent (w/w ratio) solution solution (%) 0.7 butyl acetate 0.5 0.3 0.860 0.8 decanol 0.5 0.3 1.1 60 1.0 2-Heptanone 0.5 0.4 1.1 60 1.4tri-butyl 1.0 0.2 1.2 85 phosphate 1.6 2-Heptanone 1.0 0.4 1.1 75 2.0butyl acetate 2.0 0.3 0.8 85 2.5 decanol 2.0 0.3 1.1 85 2.6 tri-butyl2.0 0.2 1.2 90 phosphate 2.7 2-Heptanone 2.0 0.4 1.1 85 3.0 butylacetate 0.5 1.5 3.1 50 4.0 tri-butyl 0.5 1.1 5.6 72 phosphate 4.42-Heptanone 0.5 1.7 5.4 61 4.6 butyl acetate 1.0 1.5 3.1 67 4.7 butylacetate 0.5 2.5 4.4 47 5.1 decanol 0.5 1.4 7.5 73 8.0 butyl acetate 1.52.1 3.9 80 8.0 tri-butyl 1.3 1.1 5.6 80 phosphate 8.0 decanol 1.7 0.84.2 80 8.0 2-Heptanone 0.7 2.0 8.4 90 8.0 tri-butyl 0.6 1.9 9.8 90phosphate 8.0 decanol 0.5 2.2 12.0 95

The experiments showed that high extraction yield values could bereached. These high extraction yield values were not limited to highisobutanol starting concentrations or to high solvent phase to aqueousphase ratios. The high extraction yield values could be observed forboth, low isobutanol starting concentrations and low proportions ofsolvent used.

Furthermore, the experiments showed that the concentration of isobutanolin the aqueous phase can be reduced to very low amounts even whenstarting with a saturated isobutanol solution. This allows recycling ofan aqueous stream into the front end of a fermentation without limitingthe alcohol productivity of the micro-organisms, since presence of highconcentrations of alcohol in the fermentation broth can adversely impactthe ability of the micro-organisms to produce alcohol.

Example 3 Enrichment of C3-C6 Alcohols from Aqueous Solutions UsingGasoline

This example illustrates the enrichment of C3-C6 alcohols from aqueoussolutions by contacting with gasoline.

Aqueous solutions of C3-C6 alcohols, including propanol (PrOH), butanol(BuOH), isobutanol (i-BuOH) and pentanol (PenOH), were prepared andmixed with gasoline until equilibrium was reached. The gasoline used wascommercial grade with a 95 octane rating. At equilibrium, the mixtureformed an upper alcohol rich phase (light 5 phase) and a lower alcohollean phase (heavy phase). Both phases were analyzed for alcohol andwater content. The distribution coefficient of the alcohol wascalculated for each experiment by dividing the alcohol concentration inthe light phase by the concentration in the heavy phase. Similarly, thedistribution coefficient for water was calculated by dividing the waterconcentration in the light phase by the water concentration in the heavyphase. The enrichment factor was calculated by dividing the distributioncoefficient of alcohol by the distribution coefficient of water. Allexperiments were carried out at 24° C. The results are reported inTables 3.1 to 3.4.

TABLE 3.1 Enrichment of 1-butanol (BuOH) by contact with gasoline at 24°C. Light phase Heavy phase Distribution composition compositioncoefficients BuOH H₂O BuOH H₂O BuOH H₂O Enrichment Wt % Wt % Wt % Wt %Da Dw factor 0.34 0.04 0.55 99.5 0.62 0.0004 >1000 1.27 0.10 1.64 98.40.77 0.001 770 7.3 0.34 3.8 96.2 1.9 0.0036 540

TABLE 3.2 Enrichment of iso-butanol (i-BuOH) by contact with gasoline at24° C. Light phase Heavy phase Distribution composition compositioncoefficients En- i-BuOH H₂O gasoline i-BuOH H₂O i-BuOH H₂O richment Wt %Wt % Wt % Wt % Wt % Da Dw factor 0.38 0.08 99.6  0.58 99.4 0.66 0.0008800 1.05 0.11 98.8  1.62 98.4 0.65 0.0011 600 1.71 0.12 98.2  2.15 97.80.80 0.0012 700 5.00 0.34 94.7  3.07 96.9 1.63 0.0035 460 6.1 0.33 93.5 3.96 96.0 1.55 0.0034 450 15.8 1.20 83.0 4.4 95.6 3.6 0.012 300 23.02.0 75.0 4.7 95.3 4.8 0.021 230 33.0 2.7 64.3 5.3 94.7 6.3 0.028 220 495.2 45.8 6.8 93.2 7.2 0.056 130 57 6.7 36.1 7.2 92.8 7.9 0.072 110 657.6 26.9 7.4 92.6 8.8 0.082 100 62 7.9 29.7 7.3 92.7 8.6 0.085 100 80.14.2 5.9 (1)   (1)  75 11.1 13.7 (1)   (1)  (1) not enough heavy phaseformed to be analyzed.

TABLE 3.3 Enrichment of Pentanol (PenOH) by contact with gasoline at 24°C. Light phase Heavy phase Distribution composition compositioncoefficients Enrich- PenOH H₂O PenOH H₂O PenOH H₂O ment Wt % Wt % Wt %Wt % Da Dw factor 1.00 0.10 0.60 99.4 1.68 0.001 >1000 4.9 0.33 1.1598.8 4.2 0.0033 >1000 17.8 1.52 2.7 97.3 6.6 0.016 400

TABLE 3.4 Enrichment of propanol (PrOH) by contact with gasoline at 24°C. PrOH in Water in PrOH in Water in light the light heavy the heavyphase phase phase phase Enrichment Wt % Wt % Wt % Wt % Da Dw factor 0.08<0.05 0.61 99.3 0.12 <0.0005 >240 0.16 <0.05 1.9 98 0.08 <0.0005 >1600.50 <0.05 5.1 94.3 0.10 <0.0005 >200

For all tested alcohols, high enrichment factors were observed bycontacting with gasoline. The alcohol to water ratio ranged from about100 to >1000 times higher in the light phase compared to the heavyphase. The light/upper phase was the alcohol rich phase in all cases.The experiments showed that high enrichment factors can be reached usinggasoline. These high enrichment factors were not limited to high alcoholstarting concentrations and high solvent phase to aqueous phase ratios,as high enrichment values could be obtained with low alcohol startingconcentrations and low amounts of gasoline.

Furthermore, in case of isobutanol, the concentration of alcohol in theaqueous phase could be reduced to very low amounts even when startingwith a saturated isobutanol solution. This allows recycling of anaqueous stream into the front end of a fermentation without limiting thealcohol productivity of the micro-organisms, since presence of highconcentrations of alcohol in the fermentation broth can adversely impactthe ability of the micro-organisms to produce alcohol.

Example 4 Enrichment of C3-C6 Alcohols from Aqueous Solutions UsingGasoline and Glucose Addition

This example illustrates the enrichment of C3-C6 alcohols from aqueoussolutions by addition of glucose followed by contacting with gasoline.Aqueous solutions of C3-C6 alcohols, including butanol (BuOH),iso-butanol (i-BuOH) and pentanol (PenOH), were prepared and glucoseadded to a final concentration of 20% (w/w). These solutions were thenmixed with gasoline. The gasoline used was commercial grade with a 95octane rating. At equilibrium, phase separation was observed forming anupper alcohol rich phase (light phase) and a lower alcohol lean phase(heavy phase). Both phases were analyzed for alcohol and water content.The distribution coefficient of the alcohol was calculated for eachexperiment by dividing the alcohol concentration in the light phase bythe concentration in the heavy phase. The distribution coefficient forwater was calculated accordingly. The Enrichment factor was calculatedby dividing the distribution coefficient of alcohol by the distributioncoefficient of water. All experiments were carried out at 24° C. Theresults are reported in Table 4.

TABLE 4 The effect of glucose on the enrichment of iso-butanol (i-BuOH)by contact with gasoline at 24° C. Light phase Heavy phase compositioncomposition Alcohol H₂O Alcohol H₂O Alcohol Alcohol Wt % Wt % Wt % Wt %Da Isobutanol 1.9 0.11 1.4 78.6 1.36 Butanol 1.9 0.11 1.5 78.5 1.27Pentanol 3.4 0.12 0.16 79.8 21

The effect of the glucose addition can be seen when comparing theseresults to the results of the experiments described in example 3. Theconcentration of the alcohol in the gasoline phase increased by 1.6 foldfor butanol, by 2 fold for isobutanol and by greater than 10 fold forpentanol in the presence of glucose. The enrichment factors increased byabout the same factors.

Example 5 Enrichment of C3-C6 Alcohols from Aqueous Solutions UsingGasoline and Calcium Chloride Addition

This example illustrates the enrichment of C3-C6 alcohols from diluteaqueous solutions by calcium chloride addition followed by contactingwith gasoline. Aqueous solutions of C3-C6 alcohols, including butanol(BuOH), isobutanol (i-BuOH) and pentanol (PenOH), were prepared andcalcium chloride was added to a final concentration of 15% (w/w). Thesesolutions were then mixed with gasoline. The gasoline used wascommercial grade with a 95 octane rating. At equilibrium, phaseseparation was observed, forming an upper alcohol rich phase (lightphase) and a lower alcohol lean phase (heavy phase). Both phases wereanalyzed for alcohol and water contents. The distribution coefficient ofthe alcohol was calculated for each experiment by dividing the alcoholconcentration in the light phase by the concentration in the heavyphase. All experiments were carried out at 24° C. The results aresummarized in Table 5.

TABLE 5 The effect of calcium chloride on the enrichment of iso-butanol(i-BuOH) by contact with gasoline at 24° C. Light phase Heavy phasecomposition composition Alcohol H₂O Alcohol H₂O Alcohol Alcohol Wt % Wt% Wt % Wt % Da Isobutanol 3.3 0.17 0.94 84.1 3.5 Butanol 2.6 0.16 0.8684.1 3.0 Pentanol 2.2 0.08 0.14 84.9 15

The effect of the calcium chloride addition can be seen when comparingthese results to the results of the experiments described in example 3.The concentration of the alcohol in the gasoline phase increased by 3fold for butanol, by 4.5 fold for isobutanol and by greater than 10 foldfor pentanol in the presence of calcium chloride. The enrichment factorsincreased by about the same factors.

Example 6 Enrichment of C3-C6 Alcohols from Saturated Aqueous SolutionsUsing Gasoline and the Addition of a Hydrophilic Compound

This example illustrates the enrichment of C3-C6 alcohols from saturatedaqueous solutions by addition of calcium chloride or glucose followed bycontacting with gasoline.

Saturated aqueous solutions of C3-C6 alcohols, including butanol (BuOH),isobutanol (i-BuOH) and pentanol (PenOH), were prepared by addition ofalcohol to water until phase separation. The heavy phase was isolatedand calcium chloride or glucose was added to it to a final concentrationof 15% (w/w) and 20% (w/w), respectively. These solutions were thenmixed with gasoline. The gasoline used was commercial grade with a 95octane rating. At equilibrium, phase separation was observed forming anupper, alcohol-rich phase (light phase) and a lower, alcohol-poor phase(heavy phase). Both phases were analyzed for alcohol and water content.The extraction yield was calculated for each experiment by dividing thealcohol amount in the solvent by the initial alcohol amount in thesolution. One stage extraction was carried out at 24° C. The results aresummarized in Table 6.

TABLE 6 The effect of glucose and calcium chloride on the enrichment ofiso-butanol (i BuOH) n-butanol (BuOH) and n-Pentanol (Pentanol) bycontact with gasoline at 24° C. Initial Final alcohol alcohol Alcoholconc. Solvent/ conc. conc. (wt %) aqueous (wt %) (wt %) The in solutionin in C3-C6 aqueous w/w aqueous solvent Extraction alcohol Additivesolution ratio solution solution yield (%) i-BuOH — 8.0 6.4 1.6 1.0 80i-BuOH 20% 8.0 3.4 1.4 1.9 80 glucose i-BuOH 15% 8.0 2.1 0.9 3.3 90CaCl2 BuOH 8.0 4.9 1.6 1.3 80 BuOH 20% 8.0 3.4 1.5 1.9 80 glucose BuOH15% 8.0 2.7 0.9 2.6 90 CaCl2 Pentanol — 2.7 2.1 0.6 1.0 75 Pentanol 20%2.7 0.8 0.16 3.4 90 glucose Pentanol 15% 2.7 1.2 0.14 2.2 90 CaCl₂

The results illustrate that higher extraction yield values could bereached if either glucose or calcium chloride were present in thesolution. Thus, a larger fraction of the alcohol can be extracted by asmaller amount of gasoline. At the same time lower concentrations of thealcohol were reached in the remaining aqueous solution.

Example 7 Phase Separation of Propanol from Aqueous Solutions byAddition of a Hydrophilic Compound

This example illustrates the induction of phase separation of propanolin water solutions by addition of a hydrophilic compound. Aqueoussolutions of propanol were prepared and hydrophilic compounds, includingcalcium chloride, sucrose or glucose, were added to various finalconcentrations. The number of liquid phases present in each mixture at25° C. was noted. The results are summarized in Table 7.

TABLE 7 Enrichment by adding hydrophilic solute to propanol solutionsHydrophilic solute Solute concentration Number of phases None 1 CaCl₂9.5%  1 CaCl₂ 15.1%   2 Glucose 40% 1 Glucose 60% 2 Sucrose 40% 2

Propanol is fully miscible with water, as it forms a single liquid phasewhen mixed with water at any proportion. This is also true if lowconcentrations of hydrophilic compounds are added to the aqueouspropanol solution. However, addition of a high enough concentration,which is dependent on the particular hydrophilic compound used, resultedin the formation of a two phase systems where propanol was highlyenriched in the light phase compared to the heavy phase.

Example 8 Phase Separation of Tertiary-Butanol (or t-Butanol) fromAqueous Solutions by Addition of Glucose

This example illustrates the induction of phase separation of t-butanol(t-BuOH) in water solution by addition of glucose. An aqueous solutionof t-butanol (50% v/v) was prepared at 25° C. and glucose was added to afinal concentration of 20% (w/v). Two liquid phases formed, where 42% ofthe volume was heavy phase and 58% light phase. Both phases wereanalyzed for t-butanol concentration. The t-BuOH and glucoseconcentrations in the heavy phase were about 150 g/L and about 45% w/w,respectively. The light phase had about 520 g/L of t-BuOH.

t-BuOH is fully miscible with water, as it forms a single phase whenmixed with water at any proportion. However, addition of glucoseresulted in the formation of two phases. t-BuOH was enriched in thelight phase as compared to the heavy phase by a factor of about 3.5.

Example 9 Phase Separation and Concentration of Sub-Saturated Butanoland Isobutanol Solutions by Addition of Glucose

This example illustrates the induction of phase separation of butanol orisobutanol in water solutions by addition of glucose. Aqueous solutionsof 7% butanol (BuOH) or isobutanol (i-BuOH) were prepared and glucosewas added to a final concentration of 20% (w/w). Two liquid phasesformed for both alcohols. The light and heavy phases were analyzed foralcohol content for each experiment. The results are reported in Table9. All experiments were carried out at 25° C. The distributioncoefficient was calculated by division of the concentration of butanolin the light phase by the concentration of butanol in the heavy phase.

TABLE 9 Enrichment by adding glucose to aqueous solutions of butanol andiso-butanol Alcohol in light phase Alcohol in heavy phase DistributionAlcohol (% w/w) (% w/w) coefficient BuOH 85 4.3 20 i-BuOH 87 5.1 17

The results of this experiment show that addition of glucose tosub-saturated solutions of butanol and isobutanol forces phaseseparation. Two liquid phases were formed where the light/upper phase isalcohol rich and the heavy/lower phase is alcohol lean (water rich).Butanol was more concentrated in the light phase by 20 fold compared tothe heavy phase and by approximately 12 fold compared to the startingsolution. Isobutanol was more concentrated in the light phase by 17 foldcompared to the heavy phase and by approximately 12 fold compared to thestarting solution.

Example 10 Phase Separation and Concentration of Sub-SaturatedIsobutanol Solutions by Addition of Glucose

This example illustrates the induction of phase separation forisobutanol in water solutions by glucose addition. Sub-saturated aqueoussolutions of isobutanol were prepared and glucose was added to a finalconcentration of 5% to 20% (w/w). Two liquid phases formed for allconcentrations. Each light and heavy phase was analyzed for isobutanolcontent. The results are reported in Table 10. All experiments werecarried out at 25° C. The distribution coefficient was calculated bydivision of the concentration of isobutanol in the light phase by theconcentration of isobutanol in the heavy phase.

TABLE 10 Light phase Heavy phase composition composition H₂O i-BuOHGlucose i-BuOH Distribution Wt % Wt % Wt % Wt % coefficient 16.1 83.95.0 8.3 10 15.1 84.9 10.6 6.8 12.5 13.7 86.2 20.4 5.7 15

The results of this experiment show that the addition of glucose tosub-saturated solutions of isobutanol forces phase separation. Twoliquid phases were formed where the light/upper phase is alcohol richand the heavy/lower phase is alcohol lean (water rich). The distributioncoefficient increased with higher concentrations of glucose.

Example 11 Phase Separation of Sub-Saturated Aqueous Butanol andIsobutanol Solutions by Addition of Calcium Chloride

This example illustrates the induction of phase separation of butanol orisobutanol in water solutions by calcium chloride addition. Aqueoussolutions of 4% butanol (BuOH) or isobutanol (i-BuOH) were prepared andcalcium chloride was added to a final concentration of 15% (w/w). Twoliquid phases formed for both alcohols. The light and heavy phases wereanalyzed for alcohol content for each experiment. The results arereported in Table 11. All experiments were carried out at 25° C. Thedistribution coefficient was calculated by division of the concentrationof butanol in the light phase by the concentration of butanol in theheavy phase.

TABLE 11 Enrichment by adding calcium chloride to aqueous solutions ofbutanol and iso-butanol Alcohol in light phase Alcohol in heavy phaseDistribution Alcohol (% w/w) (% w/w) coefficient BuOH 90 2.3 39 i-BuOH91 2.9 31

The results of this experiment show that addition of calcium chloride tosub-saturated solutions of butanol and isobutanol forces phaseseparation. Two liquid phases formed where the light/upper phase wasalcohol rich and the heavy/lower phase was alcohol lean. Butanol wasmore concentrated in the light phase by 39 fold compared to the heavyphase and by approximately 23 fold compared to the starting solution.Isobutanol was more concentrated in the light phase by 31 fold comparedto the heavy phase and by approximately 23 fold compared to the startingsolution.

Example 12 Enrichment of Butanol by Adsorption

This example illustrates the enrichment of butanol by adsorption.

The experiment was carried out using Amberlite® XAD16, which is anon-ionic, hydrophobic, cross-linked polymeric adsorbent to extractbutanol from an aqueous solution. The adsorption capacity of Amberlite®XAD16 is derived from its macromolecular structure, high surface areaand the aromatic nature of its surface.

Sub-saturated aqueous solutions of butanol were prepared and contactedwith the resin. The experiment was carried out at 25° C. The resin andthe aqueous butanol solution were mixed and shaken for 1.5 h. Aftershaking the resin was separated from the aqueous solution. Thecomposition of the separated aqueous solution, of the resin afterenrichment and the distribution coefficient are reported in Table 12.

TABLE 12 Enrichment by contact with Amberlite ® XAD16 Solutioncomposition Resin composition Distribution BuOH BuOH/resin coefficientWt % Wt % Da 2.9 30.0 10.3 1.39 21.5 15.5 0.45 10.4 23 0.11 4.0 35

The results of this experiment show high efficiency of the enrichment.Butanol was more concentrated in the resin compared to the concentrationin the aqueous solution with distribution coefficients ranging fromabout 10 to about 35.

Example 13 Enrichment of Isobutanol from Aqueous Solutions with AcidicAmine-Based Extractants

This example illustrates the efficiency of isobutanol extraction fromsub-saturated aqueous solutions using an acidic, amine-based extractant.

To reach amine concentrations of 1 mol/kg, trioctylamine was mixed withdecane. This organic solution had to be subsequently acidified withsulfuric acid. To do so, the amine solution was mixed with sulfuric acid(30% or 75% acid w/w) resulting in the extraction of parts of the acidinto the organic phase. The mixture formed two phases which wereseparated and the acidified organic phase was used as the acidicextractant for the extraction of isobutanol from an aqueous solution.The extraction was carried out by mixing the acidic extractant with theaqueous isobutanol solutions at 25° C. until equilibrium was reached.The phases were then separated and analyzed. In some cases only oneextractant phase formed, while in other cases the formation of twoextractant phases could be observed. When two extractant phases wereformed, these phases were combined and octanol, acting as a co-solvent,was added to force the formation of a single liquid phase. The combinedphase was analyzed and the results were corrected for the dilution bythe co-solvent. The aqueous phase and the extractant phase were analyzedfor sulfuric acid and isobutanol concentration. The results aresummarized in Table 13.

TABLE 13 Enrichment by contact with an acidic amine-based extractantExtractant phase Number of H₂SO₄ Aqueous phase extractant i-BuOH Moli-BuOH H₂SO₄ H₂SO₄ i-BuOH phases Wt % eq./Kg Wt % Mol eq./Kg Wt % Da 22.6 1.77 0.26 2.5 12.1 10.1 2 1.28 1.77 0.11 2.6 12.7 11.6 2 3.4 1.050.90 0.45 2.20 3.7 1 7.9 1.05 2.2 0.25 1.24 3.6 1 3.5 1.06 1.29 0.221.09 2.7 2 2.1 1.03 0.48 0.20 0.97 4.4 2 1.18 1.05 0.13 0.19 0.94 9.3

The results show large distribution coefficients for the extraction intothe acidic, amine-based extractant. The distribution coefficients weredependent on the sulfuric acid concentration in the acidic extractant.

When the concentration of sulfuric acid in the extractant phase wasabout one equivalent per mol of amine, the distribution coefficientswere similar or slightly higher compared to the distributioncoefficients determined for polar solvents, such as tri-butyl-phosphateand decanol (see Example 1). However, when the concentrations of thesulfuric acid was greater than that, very high distribution coefficients(>10) were observed.

Example 14 Enrichment of Isobutanol from Dilute Aqueous Solutions withLow Amounts Acidic Amine-Based Extractants

This example illustrates the efficiency of isobutanol enrichment fromdilute aqueous isobutanol solutions using low amounts of acidic,amine-based extractant.

To reach amine concentrations of 1 mol/kg, trioctylamine was mixed withdecane. This organic solution had to be subsequently acidified withsulfuric acid. To do so, the amine solution was mixed with sulfuric acid(30% or 75% acid w/w) resulting in the extraction of parts of the acidinto the organic phase. The mixture formed two phases which wereseparated and the acidified organic phase was used as the acidicextractant for the enrichment of isobutanol from an aqueous solution.The enrichment was carried out by mixing the acidic extractant with theaqueous isobutanol solutions at 25° C. until equilibrium was reached.The phases were then separated and analyzed. In some cases only oneextractant phase formed, while in other cases the formation of twoextractant phases could be observed. When two extractant phases wereformed, these phases were combined and octanol, acting as a co-solvent,was added to force the formation of a single liquid phase. The combinedphase was analyzed and the results were corrected for the dilution bythe co-solvent. The aqueous phase and the extractant phase were analyzedfor sulfuric acid and isobutanol concentration, the extraction yield wascalculated and the results were summarized in Table 14

TABLE 14 Enrichment of i-BuOH from aqueous solutions of variousconcentrations into acidic, amine-based extractants Initial i- H2SO4Final i- BuOH conc. concentration BuOH conc. i-BuOH (wt %) in in theSolvent/aqueous (%) in conc. (Wt %) aqueous extractant solution w/waqueous in the Extraction solution (Wt %) ratio solution extractantyield (%) 6.1 1.05 0.5 2.2 7.9 64 3.0 1.05 0.5 1.29 3.5 56 2.6 1.05 1.00.48 2.1 80 1.3 1.05 1.0 0.13 1.18 90 4.3 1.05 1.0 0.90 3.4 80 1.6 1.80.5 0.26 2.6 80 1.4 1.8 1.0 0.11 1.3 93

The results show that high extraction yield values can be reached by aone step extraction at 25° C. even for low solvent to aqueous phaseratios. Furthermore, the same high extraction yield values could bereached for low starting concentrations of isobutanol in water. Inaddition, the concentration of isobutanol in the aqueous phase could bereduced to very low concentrations for all starting concentrations. Thisallows recycling of an aqueous stream into the front end of afermentation without limiting the alcohol productivity ofmicro-organisms.

Example 15 Enrichment by Contact with Molecular Sieves

This example illustrates the enrichment of alcohol/water solutions byadsorption of water to molecular sieves.

Alcohol/water feed solutions of isopropanol, isobutanol and isopentanolwere prepared. For isopropanol, a solution containing about 6.5 wt %water was used as a feed stream. For isobutanol and isopentanol,approximately equal volumes of water and alcohol were mixed. The mixturewas allowed to separate and the water saturated alcohol phase (lightphase) was decanted and used as a feed stream. Columns consisting ofoven dried ceramic substrate, 3 Angstrom, 8×12 mesh molecular sieve bedswere prepared. The column used in the isopropanol case had a length todiameter ratio of 20. The columns used in the isobutanol and isopentanolcases had a length to diameter ratio of 40. Each feed solution wasindependently pumped from bottom to top through a separate column at aflow rate of about 10 ml/min Product samples were collected downstreamof the columns for each alcohol stream and analyzed for water contentusing a Karl Fischer Coulometer. As shown in the column ROH/H₂O below,the alcohol to water ratio (ROH/H₂O) increased in the product streamcompared to the feed stream indicating that the product stream wasenriched in alcohol. As shown in the column C_(Product)/C_(Feed), thealcohol concentration in the product stream to feed stream ratio(C_(product)/C_(Feed)) is greater than 1 indicating that the productstream was enriched in alcohol.

Enrichment Alcohol ROH/H₂O C_(product)/C_(Feed) Isopropanol Feed 14 1.1Isopropanol Product 339 Isobutanol Feed 6 1.2 Isobutanol Product 1147Isopentanol Feed 9 1.1 Isopentanol Product 2724

Example 16 Enrichment by Generation of Isobutanol and Water AzeotropeComposition

This example illustrates the enrichment of isobutanol by the generationof the isobutanol water azeotrope composition at a pressure of about 990mbar.

A 4.7% by weight solution of isobutanol in water solution was added to a250 mL round bottom flask which was equipped with a short pathdistillation head fitting, vapor temperature reading, and 25 mL overheadcondensate receiver. The 250 mL flask was heated with an electric hotplate. The isobutanol in water solution in the distillation flask washeated until vapor was formed and condensed in the overhead flask. Vaportemperature was recorded and the solution mixture was analyzed forisobutanol content. The vapor temperature of the boiling solution wasabout 89-92° C. The azeotropic vapor was collected into the overheadflask and condensed. The overhead condensed material formed a lightphase of 10.3 mL and a heavy phase of 3.1 mL. The composition of thelight phase was 670 g/L isobutanol and the heavy water rich phase was 86g/L isobutanol. The post distillation sample remaining behind in theround bottom flask contained 17 g/L isobutanol.

The example demonstrates the efficient concentration of isobutanol to670 g/L starting from a dilute solution comprising 47 g/L isobutanol bygenerating the minimum boiling point azeotropic vapor and condensing thevapor into an isobutanol rich light phase and water rich heavy phase.

Example 17 Enrichment by Generation of 1-Butanol and Water AzeotropeComposition

This example illustrates the enrichment of 1-butanol by the generationof the 1-butanol water azeotrope composition at a pressure of about 990mbar.

A 4.6% by weight solution of 1-butanol in water was added to a 250 mLround bottom flask equipped with a short path distillation head fitting,vapor temperature reading, and 25 mL overhead condensate receiver. The250 mL flask was heated with an electric hot plate. The solution in thedistillation flask was heated until vapor was formed. Vapor temperaturewas recorded and compositional analysis performed on overhead condensateand post distillation sample in the flask. The temperature of condensingvapor ranged from 92-93.5° C. Azeotropic vapor was collected into theoverhead flask and condensed. The overhead condensed material formed alight phase of 8.1 mL and a heavy phase of 2.8 mL. The composition ofthe light phase was 687 g/L 1-butanol and the heavy water rich phase was80 g/L 1-butanol. The post distillation pot sample was 20 g/L 1-butanol.

The example demonstrates the efficient concentration of 1-butanolstarting with a dilute 46 g/L composition by generating the minimumboiling point azeotrope vapor and condensing the vapor into anisobutanol rich light phase and water rich heavy phase.

Example 18 Enrichment by Generation of Iso-Butanol and WaterAzeotropecomposition

This example illustrates the enrichment of isobutanol from fermentationbroth by the generation of the isobutanol water azeotrope composition ata pressure of about 990 mbar.

A solution from a laboratory batch fermentation containing 2 g/Lisobutanol was added to a 250 mL round bottom flask which has a shortpath distillation head fitting, vapor temperature reading, and 25 mLoverhead condensate receiver. The 250 mL flask was heated with anelectric hot plate. The solution is the distillation flask was heateduntil vapor was formed and condensed in the overhead flask. Vaportemperature was recorded and compositional analysis performed onoverhead condensate and post distillation pot sample. The temperature ofcondensing vapor ranged from 89-92° C. Azeotropic vapor was condensedand collected into the overhead flask. The composition of the overheadcondensed material was 40 g/L isobutanol. The post distillation potsample was 1 g/L 1-butanol.

Example 19 Enrichment by Generation of Iso-Butanol and Water AzeotropeComposition

This example demonstrates the enrichment of isobutanol by the generationof isobutanol water azeotrope composition at a pressure of about 155mbar.

A 400 mL solution from a laboratory batch fermentation containing 6 g/Lisobutanol was added to a 1000 mL rotary vacuum evaporation apparatus.The rotary vacuum evaporation flask was heated with a hot water bath.The vaporized material was condensed with chilled water against a glasslaboratory condenser connected to a vacuum source. A vapor thermocouplein the vapor stream recorded the vapor temperature. Condensed materialwas collected in a 100 mL vacuum collection flask. In the experiment,vacuum was pulled on the system until vapor was formed. Vaportemperature was recorded and compositional analysis performed onoverhead condensate and post distillation pot sample.

The temperature of condensing vapor ranged from 27-30° C. at 155 mbarabsolute. Azeotropic vapor was condensed and collected into the overheadflask. The overhead condensed material formed a light phase of 0.26 mLand a heavy phase of 0.21 mL. The composition of the light phase was 623g/L isobutanol and the heavy water rich phase was 115 g/L 1-butanol. Thepost distillation pot sample was 3 g/L isobutanol. The exampledemonstrates the efficient concentration of starting with a dilutestarting composition and generating the minimum boiling point azeotropeto vapor. Phase separation volume split was not measurable due to smalltotal sample volume.

General methods used in Examples 20-25 and 32-33 are described below.

Sample preparation: All Samples (2 mL) from fermentation experimentsperformed in shake flasks were stored at −20° C. for later substrate andproduct analysis. Prior to analysis, samples were thawed, mixed well,and then centrifuged at 14,000×g for 10 min. The supernatant wasfiltered through a 0.2 μm filter. Analysis by HPLC or GC of substratesand products was performed using authentic standards (>99%, obtainedfrom Sigma-Aldrich), and a five-point calibration curve (with 1-pentanolas an internal standard for analysis by gas chromatography).

Determination of optical density and cell dry weight: The opticaldensity of cultures was determined at 600 nm using a DU 800spectrophotometer (Beckman-Coulter, Fullerton, Calif., USA). Sampleswere diluted as necessary to yield an optical density of between 0.1 and0.8. The cell dry weight was determined by centrifuging 50 mL of cultureprior to decanting the supernatant. The cell pellet was washed once with50 mL of milliQ H₂O, centrifuged and the pellet was washed again with 25mL of milliQ H₂O. The cell pellet was then dried at 80° C. for at least72 hours. The cell dry weight was calculated by subtracting the weightof the centrifuge tube from the weight of the centrifuge tube containingthe dried cell pellet. For E. coli cultures, an OD600 to cell dry weightconversion factor of 0.25 was used.

Gas Chromatography: Analysis of volatile organic compounds, includingethanol and isobutanol, was performed on a HP 5890 gas chromatographfitted with an HP 7673 Autosampler, a DB-FFAP column (J&W; 30 m length,0.32 mm ID, 0.25 μm film thickness) or equivalent connected to a flameionization detector (FID). The temperature program was as follows: 200°C. for the injector, 300° C. for the detector, 100° C. oven for 1minute, 70° C./minute gradient to 235° C., and then hold for 2.5 minAnalysis was performed using authentic standards (>99%, obtained fromSigma-Aldrich), and a 5-point calibration curve with 1-pentanol as theinternal standard.

High Performance Liquid Chromatography: Analysis of glucose and organicacids was performed on a HP-1100 High Performance Liquid Chromatographysystem equipped with a Aminex HPX-87H Ion Exclusion column (Bio-Rad,300×7.8 mm) or equivalent and an H⁺ cation guard column (Bio-Rad) orequivalent. Organic acids were detected using an HP-1100 UV detector(210 nm, 8 nm, 360 nm reference) while glucose was detected using anHP-1100 refractive index detector. The column temperature was 60° C.This method was isocratic with 0.008 N sulfuric acid in water as mobilephase. Flow was set at 0.6 mL/min. Injection size was 20 μL and the runtime was 30 minutes.

Molecular biology and bacterial cell culture: Standard molecular biologymethods for cloning and plasmid construction were generally used, unlessotherwise noted (Sambrook, J., Russel, D. W. Molecular Cloning, ALaboratory Manual. 3 ed. 2001, Cold Spring Harbor, N.Y.: Cold SpringHarbor Laboratory Press). Standard recombinant DNA and molecular biologytechniques used in the Examples are well known in the art and aredescribed by Sambrook, J., Russel, D. W. Molecular Cloning, A LaboratoryManual. 3 ed. 2001, Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press; and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist,Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1984) and by Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, pub. by Greene Publishing Assoc. andWiley-Interscience (1987).

General materials and methods suitable for the routine maintenance andgrowth of bacterial cultures are well known in the art. Techniquessuitable for use in the following examples may be found as set out inManual of Methods for General Bacteriology (Phillipp Gerhardt, R. G. E.Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R.Krieg and G. Briggs Phillips, eds.), American Society for Microbiology,Washington, D.C. (1994)) or by Thomas D. Brock in Biotechnology: ATextbook of Industrial Microbiology, Second Edition, Sinauer Associates,Inc., Sunderland, Mass. (1989).

Preparation of Electrocompetent Cells and Transformation: The acceptorstrain culture was grown in SOB-medium (Sambrook, J., Russel, D. W.Molecular Cloning, A Laboratory Manual. 3 ed. 2001, Cold Spring Harbor,N.Y.: Cold Spring Harbor Laboratory Press) to an OD600 of about 0.6 to0.8. The culture was concentrated 100-fold, washed once with ice coldwater and 3 times with ice cold 10% glycerol. The cells were thenresuspended in 150 μL of ice-cold 10% glycerol and aliquoted into 50 μLportions. These aliquots were used immediately for standardtransformation or stored at −80° C. These cells were transformed withthe desired plasmid(s) via electroporation. After electroporation, SOCmedium (Sambrook, J., Russel, D. W. Molecular Cloning, A LaboratoryManual. 3 ed. 2001, Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press) was immediately added to the cells. After incubationfor an hour at 37° C. the cells were plated onto LB-plates containingthe appropriate antibiotics and incubated overnight at 37° C.

Example 20 Production and Recovery of Isobutanol Using an IntegratedFermentation and Recovery System

This example illustrates the production and recovery of isobutanol usingan integrated fermentation and recovery system.

GEV01780 is a modified bacterial biocatalyst that contains genes on twoplasmids which encode a pathway of enzymes that convert pyruvate intoisobutanol. When the biocatalyst GEV01780 was contacted with glucose ina medium suitable for growth of the biocatalyst, at about 30° C., thebiocatalyst produced isobutanol from the glucose. An overnight starterculture was started in a 250 mL Erlenmeyer flask with GEV01780 cellsfrom a freezer stock with a 40 mL volume of modified M9 mediumconsisting of 85 g/L glucose, 20 g/L yeast extract, 20 μM ferriccitrate, 5.72 mg/L H₃BO₃, 3.62 mg/L MnCl₂.4H₂O, 0.444 mg/L ZnSO₄.7H₂O,0.78 mg/L Na₂MnO₄.2H₂O, 0.158 mg/L CuSO₄.5H₂O, 0.0988 mg/L CoCl₂.6H₂O,6.0 g/L NaHPO₄, 3.0 g/L KH₂PO₄, 0.5 g/L NaCl, 2.0 g/L NH₄C1, 0.0444 g/LMgSO4, and 0.00481 g/L CaCl₂ and at a culture OD₆₀₀ of about 0.05. Thestarter culture was grown for approximately 14 hrs in a 30° C. shaker at250 rpm. Some of the starter culture was then transferred to a 2000 mLDasGip fermentor vessel containing about 1500 mL of modified M9 mediumto achieve an initial culture OD₆₀₀ of about 0.1. The fermentor vesselwas attached to a computer control system to monitor and control pH at6.5 through addition of base, temperature at about 30° C., dissolvedoxygen, and agitation. The vessel was agitated, with a minimum agitationof 400 rpm and agitation was varied to maintain a dissolved oxygencontent of about 50% using a 25 sL/h air sparge until the OD₆₀₀ wasabout 1.0. The vessel was then induced with 0.1 mM IPTG. Aftercontinuing growth for approximately 8-10 hrs, the dissolved oxygencontent was decreased to 5% with 400 rpm minimum agitation and 10 sl/hairflow. Continuous measurement of the fermentor vessel off-gas by GC-MSanalysis was performed for oxygen, isobutanol, ethanol, and carbondioxide throughout the experiment. Samples were aseptically removed fromthe fermentor vessel throughout the experiment and used to measureOD₆₀₀, glucose concentration and isobutanol concentration in the broth.Throughout the experiment, supplements of pre-grown and pre-inducedbiocatalyst cells were added as a concentrate three times since thestart of the experiment: at 21 h, 38 h, and 46.3 h. These cells were thesame strain and plasmids shown above and used in the fermentor.Supplemented cells were grown as 1 L cultures in 2.8 L Fernbach flasksand incubated at 30° C., 250 RPM in Modified M9 Medium with 85 g/L ofglucose. Cultures were induced upon inoculation with 0.1 mM IPTG. Whenthe cells had reached an OD₆₀₀ of about 4.0-5.0, the culture wasconcentrated by centrifugation and then added to the fermentor. Asterile glucose feed of 500 g/L glucose in DI water was usedintermittently during the production phase of the experiment at timepoints great than 12 h to maintain glucose concentration in thefermentor of about 30 g/L or above.

The fermentor vessel was attached by tubing to a smaller 400 mLfermentor vessel that served as a flash tank and operated in arecirculation loop with the fermentor. The biocatalyst cells within thefermentor vessel were isolated from the flash tank by means of across-flow filter placed in-line with the fermentor/flash tankrecirculation loop. The filter only allowed cell-free fermentation brothto flow from the fermentor vessel into the flash tank. The volume in theflash tank was approximately 100 mL and the hydraulic retention time wasabout 10 minutes. Heat and vacuum were applied to the flash tank. Thevacuum level applied to the flash tank was initially set at 45 mBar andthe flash tank was set at about 45° C. These parameters were adjusted tomaintain approximately 6-10 g/L isobutanol in the fermentor throughoutthe experiment. Generally, the vacuum ranged from 45-100 mBar and theflash tank temperature ranged from 43° C. to 45° C. throughout theexperiment. Vapor from the heated flash tank was condensed into acollection vessel as distillate. Cell-free fermentation broth wascontinuously returned from the flash tank to the fermentation vessel.

The distillate recovered in the experiment was strongly enriched forisobutanol. Isobutanol formed an azeotrope with water and usually leadto a two phase distillate: an isobutanol rich top phase and anisobutanol lean bottom phase. Distillate samples were analyzed by GC forisobutanol concentration. Isobutanol production reached a maximum ataround 95 hrs with a batch concentration of about 63 g/L. The isobutanolproduction rate was about 0.64 g/L/h and the percent theoretical yieldwas approximately 86%.

Example 21 Production and Recovery of Isobutanol Using an IntegratedFermentation and Recovery System

This example illustrates the production and recovery of isobutanol usingan integrated fermentation and recovery system.

GEV01780 is a modified bacterial biocatalyst that contains genes on twoplasmids which encode a pathway of enzymes that convert pyruvate intoisobutanol. When the biocatalyst GEV01780 was contacted with glucose ina medium suitable for growth of the biocatalyst, at about 30° C., thebiocatalyst produced isobutanol from the glucose. An overnight starterculture was started in a 250 mL Erlenmeyer flask with GEV01780 cellsfrom a freezer stock with a 40 mL volume of modified M9 mediumconsisting of 85 g/L glucose, 20 g/L yeast extract, 20 μM ferriccitrate, 5.72 mg/L H₃BO₃, 3.62 mg/L MnCl₂.4H₂O, 0.444 mg/L ZnSO₄.7H₂O,0.78 mg/L Na₂MnO₄.2H₂O, 0.158 mg/L CuSO₄.5H₂O, 0.0988 mg/L CoCl₂.6H₂O,6.0 g/L NaHPO₄, 3.0 g/L KH₂PO₄, 0.5 g/L NaCl, 2.0 g/L NH₄Cl, 0.0444 g/LMgSO4, and 0.00481 g/L CaCl₂ and at a culture OD₆₀₀ of 0.02 to 0.05. Thestarter culture was grown for approximately 14 hrs in a 30° C. shaker at250 rpm. Some of the starter culture was then transferred to a 2000 mLDasGip fermentor vessel containing about 1500 mL of modified M9 mediumto achieve an initial culture OD₆₀₀ of about 0.1. The vessel wasattached to a computer control system to monitor and control pH at 6.5through addition of base, temperature at about 30° C., dissolved oxygen,and agitation. The vessel was agitated, with a minimum agitation of 400rpm and agitation was varied to maintain a dissolved oxygen content ofabout 50% using a 25 sL/h air sparge until the OD₆₀₀ was about 1.0. Thevessel was then induced with 0.1 mM IPTG. After continuing growth forapproximately 8-10 hrs, the dissolved oxygen content was decreased to 5%with 400 rpm minimum agitation and 10 sl/h airflow. Continuousmeasurement of the fermentor vessel off-gas by GC-MS analysis wasperformed for oxygen, isobutanol, ethanol, and carbon dioxide throughoutthe experiment. Samples were aseptically removed from the fermentorvessel throughout the experiment and used to measure OD₆₀₀, glucoseconcentration, and isobutanol concentration in the broth. Throughout theexperiment, supplements of pre-grown and pre-induced biocatalyst cellswere added as a concentrate two times after the start of the experiment:at 40 h and 75 h. These cells were the same strain and plasmids shownabove and used in the fermentor. Supplemented cells were grown as 1 Lcultures in 2.8 L Fernbach flasks and incubated at 30° C., 250 RPM inModified M9 Medium with 85 g/L glucose. Cultures were induced uponinoculation with 0.1 mM IPTG. When the cells had reached an OD₆₀₀ ofabout 4.0-5.0, the culture was concentrated by centrifugation and thenadded to the fermentor. A glucose feed of about 500 g/L glucose in DIwater was used intermittently during the production phase of theexperiment at time points greater than 12 h to maintain glucoseconcentration in the fermentor of about 30 g/L or above.

The fermentor vessel was attached by tubing to a smaller 400 mLfermentor vessel that served as a flash tank and operated in arecirculation loop with the fermentor. The biocatalyst cells within thefermentor vessel were isolated from the flash tank by means of across-flow filter placed in-line with the fermentor/flash tankrecirculation loop. The filter only allowed cell-free fermentation brothto flow from the fermentor vessel into the flash tank. The volume in theflash tank was approximately 100 mL and the hydraulic retention time wasabout 10 minutes. Heat and vacuum were applied to the flash tank. Thevacuum level applied to the flash tank was initially set at about 50mBar and the flash tank was set at about 45° C. These parameters wereadjusted to maintain approximately 6-13 g/L isobutanol in the fermentorthroughout the experiment. Generally, the vacuum ranged from 45-100 mBarand the flash tank temperature ranged from 43° C. to 45° C. throughoutthe experiment. Vapor from the heated flash tank was condensed into acollection vessel as distillate. Cell-free fermentation broth wascontinuously returned from the flash tank to the fermentation vessel.

The distillate recovered in the experiment was strongly enriched forisobutanol. Isobutanol formed an azeotrope with water and usually leadto a two phase distillate: an isobutanol rich top phase and anisobutanol lean bottom phase. Distillate samples were analyzed by GC forisobutanol concentration. Isobutanol production reached a maximum ataround 118 hrs with a batch concentration of about 87 g/L. Theisobutanol production rate was about 0.74 g/L/h on average over thecourse of the experiment. The percent theoretical yield of isobutanolwas approximately 90.4% at the end of the experiment.

Example 22 Integrated Fermentation and Recovery System for Isobutanol

This example illustrates an embodiment of an integrated fermentation andrecovery system for a C3-C6 alcohol.

GEV01780 is a modified bacterial biocatalyst that contains genes on twoplasmids which encode a pathway of enzymes that convert pyruvate intoisobutanol. When the biocatalyst GEV01780 was contacted with glucose ina medium suitable for growth of the biocatalyst, at about 30° C., thebiocatalyst produced isobutanol from the glucose. An overnight starterculture was started in a 250 mL Erlenmeyer flask with GEV01780 cellsfrom a freezer stock with a 40 mL volume of modified M9 mediumconsisting of 85 g/L glucose, 20 g/L yeast extract, 20 μM ferriccitrate, 5.72 mg/L H₃BO₃, 3.62 mg/L MnCl₂.4H₂O, 0.444 mg/L ZnSO₄.7H₂O,0.78 mg/L Na₂MnO₄.2H₂O, 0.158 mg/L CuSO₄.5H₂O, 0.0988 mg/L CoCl₂.6H₂O,6.0 g/L NaHPO₄, 3.0 g/L KH₂PO₄, 0.5 g/L NaCl, 2.0 g/L NH₄Cl, 0.0444 g/LMgSO4, and 0.00481 g/L CaCl₂ and at a culture OD₆₀₀ of about 0.05. Thestarter culture was grown for approximately 14 hrs in a 30° C. shaker at250 rpm. Some of the starter culture was then transferred to a 2000 mLDasGip fermentor vessel containing about 1500 mL of modified M9 mediumto achieve an initial culture OD₆₀₀ of about 0.1. The vessel wasattached to a computer control system to monitor and control pH at 6.5through addition of base, temperature at about 30° C., dissolved oxygen,and agitation. The vessel was agitated, with a minimum agitation of 400rpm and agitation was varied to maintain a dissolved oxygen content ofabout 50% using a 25 sL/h air sparge until the OD₆₀₀ was about 1.0. Thevessel was then induced with 0.1 mM IPTG. After continuing growth forapproximately 8-10 hrs, the dissolved oxygen content was decreased to 5%with 400 rpm minimum agitation and 10 sl/h airflow. Continuousmeasurement of the fermentor vessel off-gas by GC-MS analysis wasperformed for oxygen, isobutanol, ethanol, and carbon dioxide throughoutthe experiment. Samples were aseptically removed from the fermentorvessel throughout the experiment and used to measure OD₆₀₀, glucoseconcentration, and isobutanol concentration in the broth. Throughout theexperiment, supplements of pre-grown and pre-induced biocatalyst cellswere added as a concentrate after the start of the experiment: at 62.5h, 87 h, 113 h, and 142 h. These cells were the same strain and plasmidsshown above and used in the fermentor. Supplemented cells were grown as1 L cultures in 2.8 L Fernbach flasks and incubated at 30° C., 250 RPMin Modified M9 Medium. Cultures were induced upon inoculation with 0.1mM IPTG. When the cells had reached an OD₆₀₀ of about 4.0-5.0, theculture was concentrated by centrifugation and then added to thefermentor. A glucose feed of about 500 g/L glucose in DI water was usedintermittently during the production phase of the experiment at timepoints greater than 12 h to maintain glucose concentration in thefermentor of about 30 g/L or above.

The fermentor vessel was attached by tubing to a smaller 400 mLfermentor vessel that served as a flash tank and operated in arecirculation loop with the fermentor. The volume in the flash tank wasapproximately 100 mL and the hydraulic retention time was about 5-10minutes. Heat and vacuum were applied to the flash tank. The vacuumlevel applied to the flash tank was initially set at about 40 mBar andthe flash tank was set at about 36° C. These parameters were adjusted tomaintain approximately 5-10 g/L isobutanol in the fermentor throughoutthe experiment. Generally, the vacuum ranged from about 20-50 mBar andthe flash tank temperature of about 36° C. throughout the experiment.Vapor from the heated flash tank was condensed into a collection vesselas distillate. The fermentation broth was continuously returned from theflash tank to the fermentation vessel.

The distillate recovered in the experiment was strongly enriched forisobutanol. Isobutanol formed an azeotrope with water and led to a twophase distillate: an isobutanol rich top phase and an isobutanol leanbottom phase. Distillate samples were analyzed by GC for isobutanolconcentration. Isobutanol production reached a maximum at around 166 hrswith a batch concentration of about 106 g/L. The isobutanol productionrate was about 0.64 g/L/h and the percent theoretical yield wasapproximately 91% at the end of the experiment.

Example 23 Production and Recovery of Isobutanol Using an IntegratedFermentation and Recovery System

This example illustrates the production and recovery of isobutanol usingan integrated fermentation and recovery system.

GEV01780 is a modified bacterial biocatalyst that contains genes on twoplasmids which encode a pathway of enzymes that convert pyruvate intoisobutanol. When the biocatalyst GEV01780 was contacted with glucose ina medium suitable for growth of the biocatalyst, at about 30° C., thebiocatalyst produced isobutanol from the glucose. Overnight startercultures were started in four 2.8 L Fernbach flasks with GEV01780 cellsfrom freezer stocks with four 1000 mL volumes of modified M9 mediumconsisting of 85 g/L glucose, 20 g/L yeast extract, 20 μM ferriccitrate, 5.72 mg/L H₃BO₃, 3.62 mg/L MnCl₂.4H₂O, 0.444 mg/L ZnSO₄.7H₂O,0.78 mg/L Na₂MnO₄.2H₂O, 0.158 mg/L CuSO₄.5H₂O, 0.0988 mg/L CoCl₂.6H₂O,6.0 g/L NaHPO₄, 3.0 g/L KH₂PO₄, 0.5 g/L NaCl, 2.0 g/L NH₄Cl, 0.0444 g/LMgSO4, and 0.00481 g/L CaCl₂ and at a culture OD₆₀₀ of about 0.05. Thecultures were induced with 1 mM IPTG at the point of inoculation andgrown for approximately 14 hrs in a 30° C. shaker at 250 rpm. At about14 hours, the contents of the flasks were then poured into 500 mLsterile graduated plastic bottles and centrifuged for 20 minutes at 4500rpm. The cells were resuspended in about 100 mL total volume of modifiedM9 medium without glucose, then transferred to a 2000 mL DasGipfermentor vessel containing about 1500 mL of modified M9 medium, whereinthe glucose was replaced by clarified corn liquefact to give anapproximate glucose concentration of about 100 g/L and to achieve aninitial culture OD₆₀₀ of about 10. Clarified corn liquefact was preparedby incubating a slurry of ground corn at about 60° C. for about 24 hrsto which alpha-amylase and gluco-amalyase enzymes had been added insufficient amounts to liberate free glucose from the corn starch. Afterabout 24 hours of treatment as described above, the corn liquefact wasclarified by centrifugation and filtration to remove most of the solidsand generate a clarified corn liquefact solution of about 250 g/Lglucose. The fermentor vessel was attached to a computer control systemto monitor and control pH at 6.5 through addition of base, temperatureat about 30° C., dissolved oxygen, and agitation. The vessel wasagitated, with a minimum agitation of 400 rpm and agitation was variedto maintain a dissolved oxygen content of about 5% using a 10 sL/h airsparge. Continuous measurement of the fermentor vessel off-gas by GC-MSanalysis was performed for oxygen, isobutanol, ethanol, and carbondioxide throughout the experiment. Samples were aseptically removed fromthe fermentor vessel throughout the experiment and used to measureOD₆₀₀, glucose concentration, and isobutanol concentration in the broth.Supplements of pre-grown and pre-induced biocatalyst cells were added asa concentrate throughout this experiment. These cells were the samestrain and plasmids shown above and used in the fermentor. Supplementedcells were grown as 1 L cultures in 2.8 L Fernbach flasks and incubatedat 30° C., 250 RPM in Modified M9 Medium using glucose as the maincarbon source. Cultures were induced upon inoculation with 1 mM IPTG.When the cells had reached an OD₆₀₀ of about 2.0-5.0, the culture wasconcentrated by centrifugation and then added to the fermentor. A feedof clarified corn liquefact containing about 250 g/L glucose was usedintermittently during the experiment to maintain glucose concentrationin the fermentor of about 30 g/L or above.

The fermentor vessel was attached by tubing to a smaller 400 mLfermentor vessel that served as a flash tank and operated in arecirculation loop with the fermentor. The volume in the flash tank wasapproximately 100 mL and the hydraulic retention time was about 5-10minutes. Heat and vacuum were applied to the flash tank. The vacuumlevel applied to the flash tank was initially set at about 40 mBar andthe flash tank was set at about 36° C. These parameters were adjusted tomaintain approximately 5-10 g/L isobutanol in the fermentor throughoutthe experiment. Generally, the vacuum ranged from about 20-50 mBar andthe flash tank temperature of about 36° C. throughout the experiment.Vapor from the heated flash tank was condensed into a collection vesselas distillate. The fermentation broth was continuously returned from theflash tank to the fermentation vessel.

The distillate recovered in the experiment was strongly enriched forisobutanol. Isobutanol formed an azeotrope with water and led to a twophase distillate: an isobutanol rich top phase and an isobutanol leanbottom phase. Distillate samples were analyzed by GC for isobutanolconcentration. Isobutanol production reached a maximum at around 217 hrswith a batch concentration of about 124 g/L. The isobutanol productionrate was about 0.57 g/L/h on average over the course of the experiment,but a maximum isobutanol production rate of about 1.3 g/L/h was achievedin the experiment. The percent theoretical yield was approximately 74%at the end of the experiment, but a maximum theoretical yield of about88% theoretical yield was achieved during the experiment.

Example 24 Production and Recovery of Isobutanol Using an IntegratedFermentation and Recovery System

This example illustrates the production and recovery of isobutanol usingan integrated fermentation and recovery system.

GEV01780 is a modified bacterial biocatalyst that contains genes on twoplasmids which encode a pathway of enzymes that convert pyruvate intoisobutanol. When the biocatalyst GEV01780 was contacted with glucose ina medium suitable for growth of the biocatalyst, at about 30° C., thebiocatalyst produced isobutanol from the glucose. An overnight starterculture was started in a 2.8 L Fernbach flask with GEV01780 cells from afreezer stock with a 1000 mL volume of modified M9 medium consisting of85 g/L glucose, 20 g/L yeast extract, 20 μM ferric citrate, 5.72 mg/LH₃BO₃, 3.62 mg/L MnCl₂.4H₂O, 0.444 mg/L ZnSO₄.7H₂O, 0.78 mg/LNa₂MnO₄.2H₂O, 0.158 mg/L CuSO₄.5H₂O, 0.0988 mg/L CoCl₂.6H₂O, 6.0 g/LNaHPO₄, 3.0 g/L KH₂PO₄, 0.5 g/L NaCl, 2.0 g/L NH₄Cl, 0.0444 g/L MgSO4,and 0.00481 g/L CaCl₂ and at a culture OD₆₀₀ of about 0.05. The culturewas induced with 1 mM IPTG at the point of inoculation and grown forapproximately 14 hrs in a 30° C. shaker at 250 rpm. At about 14 hours,the contents of the flask was then poured into 500 ml sterile graduatedplastic bottles and centrifuged for 20 minutes at 4500 rpm. The cellswere resuspended in about 40 ml total volume of modified M9 medium, thentransferred to a 2000 mL DasGip fermentor vessel containing about 1500mL of modified M9 medium, wherein the glucose was replaced by cornliquefact with about 17% dry solids concentration and to achieve aninitial calculated culture OD₆₀₀ of about 3. Corn liquefact, which wastreated with alpha-amyalse, was prepared by diluting sterilized cornliquefact with a dry solids concentration of about 35% with steriledionized water to a final dry solids concentration of about 17%. Thediluted corn liquefact was then added to the modified M9 mediumcomponents described above without additional glucose and placed in the2000 mL fermentor vessel. At the point of inoculation, a dose ofgluco-amylase was added to the fermentor vessel in sufficient quantityto hydrolyse the corn starch oligomers present in the corn liquefact tomonomeric glucose. The vessel was attached to a computer control systemto monitor and control pH at about 6.5 through addition of base,temperature at about 30° C., dissolved oxygen, and agitation. The vesselwas agitated, with a minimum agitation of 400 rpm and agitation wasvaried to maintain a dissolved oxygen content of about 5% using a 10sL/h air sparge. Continuous measurement of the fermentor vessel off-gasby GC-MS analysis was performed for oxygen, isobutanol, ethanol, andcarbon dioxide throughout the experiment. Samples were asepticallyremoved from the fermentor vessel throughout the experiment and used tomeasure glucose concentration and isobutanol concentration in the broth.Supplements of pre-grown and pre-induced biocatalyst cells were added asa concentrate throughout this experiment. These cells were the samestrain shown above and used in the fermentor. Supplemented cells weregrown as 1 L cultures in 2.8 L Fernbach flasks and incubated at 30° C.,250 RPM in Modified M9 Medium using glucose as the main carbon source.Cultures were induced upon inoculation with 1 mM IPTG. When the cellshad reached an OD₆₀₀ of about 2.0-5.0, the culture was concentrated bycentrifugation and then added to the fermentor. A feed of corn liquefactwas prepared by adding dose of gluco-amylase in sufficient quantity tohydrolyse the corn starch oligomers present in the corn liquefact tomonomeric glucose and incubation at about 50° C. for 24 hrs prior touse. The resulting solution contained about 188 g/L glucose and was usedintermittently during the experiment to maintain glucose concentrationin the fermentor of about 40 g/L or above.

The fermentor vessel was attached by tubing to a smaller 400 mLfermentor vessel that served as a flash tank and operated in arecirculation loop with the fermentor. The volume in the flash tank wasapproximately 100 mL and the hydraulic retention time was about 5-10minutes. Heat and vacuum were applied to the flash tank. The vacuumlevel applied to the flash tank was initially set at about 40 mBar andthe flash tank was set at about 36° C. These parameters were adjusted tomaintain approximately 5-10 g/L isobutanol in the fermentor throughoutthe experiment. Generally, the vacuum ranged from about 20-50 mBar andthe flash tank temperature was about 36° C. throughout the experiment.Vapor from the heated flash tank was condensed into a collection vesselas distillate. The fermentation broth was continuously returned from theflash tank to the fermentation vessel.

The distillate recovered in the experiment was strongly enriched forisobutanol. Isobutanol formed an azeotrope with water and led to a twophase distillate: an isobutanol rich top phase and an isobutanol leanbottom phase. Distillate samples were analyzed by GC for isobutanolconcentration. Isobutanol production reached a maximum at around 166 hrswith a batch concentration of about 30 g/L. The isobutanol productionrate was about 0.31 g/L/h on average over the course of the experiment.The percent theoretical yield was not determined in this experiment.

Example 25 Integrated Fermentation and Recovery System for Isobutanol

This example illustrates an embodiment of an integrated anaerobic batchfermentation and recovery system for a C3-C6 alcohol and shows that anengineered microorganism produces a C3-C6 alcohol at a yield of atgreater than about 95% of theoretical.

An overnight culture was started in a 250 mL Erlenmeyer flask withGEV01886 cells from a freezer stock with a 40 mL volume of modified M9medium consisting of 85 g/L glucose, 20 g/L yeast extract, 20 μM ferriccitrate, 5.72 mg/L H₃BO₃, 3.62 mg/L MnCl₂.4H₂O, 0.444 mg/L ZnSO₄.7H₂O,0.78 mg/L Na₂MnO₄.2H₂O, 0.158 mg/L CuSO₄.5H₂O, 0.0988 mg/L CoCl₂.6H₂O,6.0 g/L NaHPO₄, 3.0 g/L KH₂PO₄, 0.5 g/L NaCl, 2.0 g/L NH₄Cl, 0.0444 g/LMgSO₄, and 0.00481 g/L CaCl₂ and at a culture OD₆₀₀ of about 0.05. Thestarter culture was grown for approximately 14 hrs in a 30° C. shaker at250 rpm. Some of the starter culture was transferred to a 2000 mL DasGipfermentor vessel containing about 1500 mL of modified M9 medium toachieve an initial culture OD₆₀₀ of about 0.1. The fermentor vessel wasattached to a computer control system to monitor and control pH at 6.5through addition of base, temperature at about 30° C., dissolved oxygen,and agitation. The vessel was agitated, with a minimum agitation of 400rpm and agitation was varied to maintain a dissolved oxygen content ofabout 50% using a 25 sL/h air sparge until the OD₆₀₀ is about 1.0. Thevessel was then induced with 0.1 mM IPTG. After continuing growth forabout 3 hours, the dissolved oxygen content was decreased to 0% with 200rpm agitation and 2.5 sL/h sparge with nitrogen (N₂) gas. Continuousmeasurement of the fermentor vessel off-gas by GC-MS analysis wasperformed for, isobutanol, ethanol, and carbon dioxide throughout theexperiment. Samples were aseptically removed from the fermentor vesselthroughout the experiment and used to measure OD₆₀₀, glucoseconcentration and isobutanol concentration in the broth. Throughout theexperiment, supplements of pre-grown and pre-induced biocatalyst cellswere added as a concentrate several times since the start of theexperiment. These cells were the same strain and plasmids shown aboveand used in the fermentor. Supplemented cells were grown as 1 L culturesin 2.8 L Fernbach flasks and incubated at 30° C., 250 RPM in Modified M9Medium with 85 g/L of glucose. Cultures were induced upon inoculationwith 0.1 mM IPTG. When the cells reached an OD₆₀₀ of about 4.0-5.0, theculture was concentrated by centrifugation and then added to thefermentor. A sterile glucose feed of 500 g/L glucose in DI water wasused intermittently during the production phase of the experiment attime points greater than 12 h to maintain glucose concentration in thefermentor of about 30 g/L or above.

The fermentor vessel was attached by tubing to a smaller 400 mLfermentor vessel that serves as a flash tank and is operated in arecirculation loop with the fermentor. The volume in the flash tank wasapproximately 100 mL and the hydraulic retention time is about 10minutes. Heat and vacuum were applied to the flash tank. The vacuumlevel applied to the flash tank was initially set at about 45 mBar andthe flash tank was set at about 36° C. These parameters were adjusted tomaintain approximately 6-10 g/L isobutanol in the fermentor throughoutthe experiment. Generally, the vacuum ranged from about 30-100 mBar andthe flash tank temperature ranged from 34° C. to 36° C. throughout theexperiment. Vapor from the heated flash tank was condensed into acollection vessel as distillate. The fermentation broth was continuouslyreturned from the flash tank to the fermentation vessel.

The distillate recovered in the experiment was strongly enriched forisobutanol. Isobutanol formed an azeotrope with water and led to a twophase distillate: an isobutanol rich top phase and an isobutanol leanbottom phase. Distillate samples were analyzed by GC for isobutanolconcentration. Isobutanol production reached a maximum with a batchconcentration of greater than 50 g/L. The percent theoretical yield wasapproximately 95%.

Example 26 Enrichment by Vacuum Distillation

This example illustrates the enrichment of unsaturated, aqueousisobutanol solutions by vacuum distillation.

The distillation set-up consisted of a 3 neck reaction vessel, a watercooled condenser and a collection flask. During each distillation, anelectric heating mantle was used to heat the reaction vessel; arecirculating bath was used to cool the condenser with 3-5° C. water;and a vacuum pump maintained a vacuum of approximately 50 mBar absolute.Thermometers were in place to measure the temperature of the reactionsolution and the water bath. The collection flask and reaction vesselwere sampled after a target volume of 1-5 mL was collected. Threedistillations were performed with aqueous isobutanol solutions ofapproximately 5, 15 and 40 g/L.

During distillation of the 5 g/L solution, condensation began at aliquid temperature of 35.2° C. 1.75 ml of single phase material wascollected. The 4.1 g/L starting material was concentrated 21-fold in theoverhead condensed vapor phase material. During distillation of the g/Lsolution, condensation began at a liquid temperature of 34.3° C. 2 mL ofheavy phase and 0.5 mL of light phase were collected. The 12.3 g/Lstarting material was concentrated 17-fold in the overhead condensedvapor phase material. During distillation of the 40 g/L solution,condensation began at a liquid temperature of 31.9° C. 2.5 mL of heavyphase and 3.5 mL of light phase were collected. The 43.2 g/L startingmaterial was concentrated 10-fold in the overhead condensed vapor phasematerial. The isobutanol concentration (g/L) from various solutionsduring the 3 isobutanol distillations are summarized in the table below.

TABLE 26 Isobutanol Distillations 5 g/L 15 g/L 40 g/L Starting Solution4.1 12.3 43.2 Final Pot 3.5 10.7 36.8 Collection: Heavy 86.7  86.9 91.9Collection: Light — 717.8 706.6The increase in isobutanol concentration from the starting solution tothe vapor phase is shown in the table below.

Isobutanol Distillations 5 g/L 15 g/L 40 g/L Concentration in Vapor 21x17x 10x Phase

Example 27 Enrichment by Vacuum Distillation

This example illustrates the enrichment of unsaturated, aqueousisopentanol solutions by vacuum distillation.

The distillation set-up consisted of a 3 neck reaction vessel, watercooled condenser and collection flask. During each distillation, anelectric heating mantle was used to heat the reaction vessel, arecirculating bath was used to cool the condenser with 3-5° C. water anda vacuum pump maintained a vacuum of approximately 50 mBar absolute.Thermometers were in place to measure reaction solution temperature andwater bath temperature. The collection flask and reaction vessel weresampled after a target volume of at least 1-5 mL was collected. Threedistillations were performed with aqueous isopentanol solutions ofapproximately 5, 15 and 40 g/L.

During distillation of the 5 g/L solution, condensation began at aliquid temperature of 35.2° C. 2.5 mL of single phase material werecollected. The 4.2 g/L starting material was concentrated 7-fold in theoverhead condensed vapor phase material. During distillation of the 15g/L solution, condensation began at a liquid temperature of 34.3° C. 3mL of heavy phase and 1 mL of light phase were collected. The 12.2 g/Lstarting material was concentrated 17-fold in the overhead condensedvapor phase material. During distillation of the 40 g/L solution,condensation began at a liquid temperature of 33.5° C. 5 mL of heavyphase and 3 mL of light phase were collected. The 31.5 g/L startingmaterial was concentrated 10-fold in the overhead condensed vapor phasematerial. The isopentanol concentration (g/L) from various solutionsduring the 3 isopentanol distillations are summarized in the tablebelow.

Isopentanol Distillations 5 g/L 15 g/L 40 g/L Starting Solution 4.2 12.231.5 Final Pot 3.4 10.2 23.7 Collection: Heavy 31.2  34.1 35.9Collection: Light — 724.7 763.7

The increase in isopentanol concentration from the starting solution tothe vapor phase are shown in the table below.

Isopentanol Distillations 5 g/L 15 g/L 40 g/L Concentration in the 7x17x 10x Vapor Phase

Example 28 Enrichment by Vacuum Distillation

This example illustrates the enrichment of aqueous isopropanol solutionsby vacuum distillation.

The distillation set-up consisted of a 3 neck reaction vessel, watercooled condenser and collection flask. During each distillation, anelectric heating mantle was used to heat the reaction vessel, arecirculating bath was used to cool the condenser with 3-5° C. water anda vacuum pump maintained a vacuum of approximately 50 mBar. Thermometerswere in place to measure reaction solution temperature and water bathtemperature. The collection flask and reaction vessel were sampled aftera target volume of at least 1-5 ml was collected. Three distillationswere performed with aqueous isopropanol solutions of approximately 5, 15and 40 g/L.

During distillation of the 5 g/L solution, condensation began at aliquid temperature of 35.0° C. 3.3 mL of single phase material werecollected. The 5.0 g/L starting material was concentrated 25-fold in theoverhead condensed vapor phase material. During distillation of the g/Lsolution, condensation began at a liquid temperature of 34.7° C. 5 mL ofsingle phase material were collected. The 14.9 g/L starting material wasconcentrated 12-fold in the overhead condensed vapor phase material. Forthe 40 g/L starting solution, condensation began at a liquid temperatureof 33.3° C. 8.1 mL of single phase material were collected. The 49.2 g/Lstarting material was concentrated 7-fold in the overhead condensedvapor phase material.

The isopropanol concentration (g/L) from various solutions during the 3isopropanol distillations are shown in the table below.

Isopropanol Distillations 5 g/L 15 g/L 40 g/L Starting Solution 5.0 14.949.2 Final Pot 3.8 12.3 31.9 Collection: Heavy 126.3  184.2  347.5 Collection: Light — — —

The increase in isopropanol concentration from the starting solution tothe vapor phase is shown in the table below.

Isopropanol Distillations 5 g/L 15 g/L 40 g/L Concentration in the 25x12x 7x Vapor Phase

Example 29 Enrichment by Vacuum Distillation

This example illustrates the enrichment of unsaturated, aqueousisobutanol by distillation.

A glass distillation set-up with a packed bead reflux column was used.The distillation was conducted in two parts, the vacuum was removed andpot cooled between parts. During the distillation, a heating mantle wasused to heat the reaction pot, an agitator kept the reaction pot wellmixed, a recirculating bath was used to cool the condenser, a vacuumpump maintained a vacuum of approximately 75 mBar absolute in the systemand an alcohol/dry ice trap was used downstream from the condenser.Thermometers were in place to measure reaction pot temperature, vaportemperature above the reflux column and water bath temperature.

During the distillations, a sample valve allowed collection from abovethe reflux column or redirection of condensate flow through a splitteronto the reflux column. Material was collected when good reflux wasobserved and the entire column was active. The starting material was asingle phase isobutanol solution collected by vacuum evaporation from a400 L scale fermentation of GEV01780. Condensation was observed on thebottom of the condenser coils as soon as the vacuum was generated with adistillation pot temperature of 22° C. and a vapor temperature of 16° C.When good reflux was observed at a pot temperature of 31° C. and a vaportemperature of 20° C. the distillate collection was initiated. Aftercollection of 840 mL, the distillation was interrupted and thedistillation pot was sampled. The isobutanol in the distillation pot wasreduced to a concentration of 17 g/L which is 27.9% of the initialamount.

During the second part of the distillation, condensation was alsoobserved as soon as the vacuum was generated. A maximum reaction pottemperature of 40° C. and vapor temperature of 26° C. was reached. Theseconditions further decreased the reaction pot isobutanol concentrationfrom 17 g/L to 1.5 g/L. At the end of the distillation, 62.2% of theisobutanol initially in the distillation pot was collected as lightphase distillate and 14.3% as heavy phase distillate, 15.4% wascollected as light phase in the vacuum ice trap and 2.7% as heavy phasein the vacuum ice trap and 2.2% was left behind in the distillation pot.Overall, the concentration increase from the starting material to thevapor phase was 5.0 fold.

Example 30 Removal of Water from the Alcohol-Rich Phase

This example illustrates the effective removal of water from the alcoholrich phase (light phase) by distillation.

A glass distillation set-up with a packed bead reflux column was used.The distillation was conducted in two parts, the vacuum was removed andpot cooled between parts. During the distillation, a heating mantle wasused to heat the reaction pot, a recirculating bath was used to cool thecondenser and agitator, a vacuum pump maintained a vacuum ofapproximately 75 mBar absolute in the system and an alcohol/dry ice trapwas used downstream from the condenser. Thermometers were in place tomeasure reaction pot temperature, vapor temperature above the refluxcolumn and water bath temperature. During the distillations, a samplevalve allowed collection from above the reflux column or redirection ofcondensate flow through a splitter onto the reflux column Material wascollected when good reflux was observed and the entire column wasactive.

The light phase (alcohol-rich phase) from 3 different Gevo fermentationswas combined. Good reflux was observed at a reaction pot temperature of27° C. and a vapor temperature of 18.5° C., collection was initiated atthis point. After collection of 580 mL, the distillation wasinterrupted. The distillation pot liquid was 1.44 wt % water at thispoint. Distillation was resumed and after reaching a maximum liquidtemperature of 48° C. and a vapor temperature of 36.5° C., thedistillation was ended. The final reaction pot solution was dried to0.3671 wt % water. The wt % water in the distillation pot was decreased40-fold.

Example 31 Impact of Temperature on Light and Heavy Phase Composition

This example illustrates the mutual solubility of isobutanol and wateras a function of temperature.

First, 20 g of water were mixed with 20 g isobutanol in closed vials atroom temperature. The vials were then mixed at 5, 10, 20, 40, 60 or 80°C. The vials were held at the stated temperature while being mixed untilequilibrium was established. Then the samples were separated into lightand heavy phases while being held at temperature. The samples from thelight and the heavy phases were analyzed by HPLC and by Karl-Fischertitration. The results are presented in Table 31.1 and show that theisobutanol concentrations in each phase are temperature dependent.

Table 31.2 summarizes literature data for normal butanol (n-butanol)solubility in water at various temperatures. As expected the solubilityof isobutanol is similar to n-butanol and shows similar dependence ontemperature.

TABLE 31.1 Light and Heavy Phase Composition of Isobutanol-Water atVarious Temperatures Light phase Heavy phase Temperature Isobutanol H₂OIsobutanol H₂O ° C. Wt % Wt % Wt % Wt % 5 83.9 16.1 11.7 88.3 10 84.016.0 10.6 89.4 20 83.4 16.6 11.2 88.8 40 81.5 18.5 9.24 90.8 60 79.120.9 7.90 92.1 80 76.5 23.5 6.04 94.0

TABLE 31.2 n-Butanol Solubility in Water at Various Temperatures heavyLight phase phase Temperature Butanol Butanol ° C. Wt % Wt % 5 9.5580.38 10 8.91 80.33 15 8.21 80.14 20 7.81 79.93 25 7.35 79.73 30 7.0879.38 35 6.83 78.94 40 6.6 78.59 50 6.46 77.58 60 5.62 76.38 70 6.7374.79 80 6.89 73.53

Example 32 High Volumetric Productivity Batch Fermentation

This example illustrates a batch fermentation using a biocatalyst withhigh volumetric productivity.

Gevo 1780 is a modified bacterial biocatalyst that contains genes on twoplasmids which encode a pathway of enzymes that convert pyruvate intoisobutanol. When the biocatalyst Gevo 1780 was contacted with glucose ina medium suitable for growth of the biocatalyst, at about 30° C., thebiocatalyst produced isobutanol from the glucose. Two 400 mL DasGipfermentor vessels containing 200 mL each of modified M9 mediumconsisting of 85 g/L glucose, 20 g/L yeast extract, 20 μM ferriccitrate, 5.72 mg/L H₃BO₃, 3.62 mg/L MnCl₂.4H₂O, 0.444 mg/L ZnSO₄.7H₂O,0.78 mg/L Na₂MnO₄.2H₂O, 0.158 mg/L CuSO₄.5H₂O, 0.0988 mg/L CoCl₂.6H₂O,6.0 g/L NaHPO₄, 3.0 g/L KH₂PO₄, 0.5 g/L NaCl, 2.0 g/L NH₄Cl, 0.0444 g/LMgSO4, and 0.00481 g/L CaCl₂ were inoculated with GEV01780 cells fromfrozen stocks. The fermentor vessels were attached to a computer controlsystem to monitor and control pH at 6.5 through addition of base,temperature at 30° C., dissolved oxygen, and agitation. The vessels wereagitated, with a minimum agitation of 300 rpm and agitation was variedto maintain a dissolved oxygen content of about 50% using a 12 sL/h airsparge until the OD₆₀₀ was about 1.0. The vessels were then induced with0.1 mM IPTG. The vessels were operated under these conditions for about12 hours. At about 12 hours, the contents of the fermentor vessels werethen poured into 500 ml sterile graduated plastic bottles andcentrifuged for 20 minutes at 4500 rpm. The cells were resuspended in 50ml total volume of modified M9 medium. A 400 mL DasGip vessel containing150 mL of modified M9 medium was inoculated with 50 ml of the cellcontaining medium and then induced with 0.1 mM IPTG. Constant dissolvedoxygen content of 5% was maintained using a 2.5 sL/h air sparge withvariable agitation automatically controlled from 300 to 1200 rpm.Continuous measurement of the fermentor vessel off gas by GC-MS analysiswas performed for oxygen, isobutanol, ethanol, carbon dioxide, andnitrogen throughout the experiment. Samples were aseptically removedfrom the fermentor vessel throughout the experiment and used to measureOD₆₀₀, glucose concentration by HPLC, and isobutanol concentration inthe broth by GC. Isobutanol production reached a maximum at around 22hours with a batch concentration of about 22 g/L and with a yield ofapproximately 80% maximum theoretical. Volumetric productivity of thefermentation, calculated when the isobutanol was between 1 g/L and 15g/L, was about 2.3 g/L/h.

Example 33 High Volumetric Productivity Batch Fermentation

This example illustrates a batch fermentation using a biocatalyst withhigh volumetric productivity. The modified biocatalyst Gevo 1530 wastransformed with the two plasmids pSA69 and pSA55, which encode apathway of enzymes that convert pyruvate into isobutanol. When thebiocatalyst Gevo 1530 (pSA69, pSA55) was contacted with glucose in amedium suitable for growth of the biocatalyst, at about 30° C., thebiocatalyst produced isobutanol from the glucose. Two 400 mL DasGipfermentor vessels containing 200 mL each of EZ Rich medium (Neidhardt,F. C., P. L. Bloch, and D. F. Smith. 1974. Culture medium forenterobacteria. Bacteriol. 119:736-47) containing 72 g/L glucose and 10g/L yeast extract were inoculated with Gevo 1530 (pSA69, pSA55) cells.The vessels were attached to a computer control system to monitor andcontrol pH at 6.5 through addition of base, temperature at about 30° C.,dissolved oxygen, and agitation. The vessels were agitated, with aminimum agitation of 300 rpm and agitation was varied to maintain adissolved oxygen content of about 50% using a 12 sL/h air sparge untilthe OD₆₀₀ was about 1.0. The vessels were then induced with 0.1 mM IPTG.The vessels were operated under these conditions for about 11 hours. Atabout 11 hours, the contents of the fermentor vessels were then pouredinto 500 mL sterile graduated plastic bottles and centrifuged for 20minutes at 4500 rpm. The cells were resuspended in 50 ml total volume ofmodified M9 medium. A 400 mL DasGip vessel containing 150 ml of EZ Richmedium containing 72 g/L glucose and 10 g/L yeast extract was inoculatedwith 50 mL of the cell containing medium and then induced with 0.1 mMIPTG. Cell concentration was approximately 6 g CDW per L. Constantdissolved oxygen content of 5% was maintained using a 2.5 sL/h airsparge with variable agitation automatically controlled from 300 to 1200rpm. Measurement of the fermentor vessel off-gas by trapping in anoctanol bubble trap and then measurement by GC was performed forisobutanol and ethanol. Continuous measurement of off-gas concentrationsof carbon dioxide and oxygen were also measured by a DasGip off-gasanalyzer throughout the experiment. Samples were aseptically removedfrom the fermentor vessel throughout the experiment and used to measurePD₆₀₀, glucose concentration by HPLC, and isobutanol concentration inthe broth by GC. Isobutanol production reached a maximum at around 4hours with a batch concentration of 15 g/L and with a yield ofapproximately 86% maximum theoretical. Volumetric productivity of thefermentation, calculated from the inception of the fermentation at time0 h to an elapsed fermentation time of about 4 h, was about 3.5 g/L/h.

Example 34 Enrichment by Contact with a Water Adsorbent

This example illustrates the isobutanol enrichment from aqueousisobutanol solutions using molecular sieves to adsorb water. Asub-saturated solution of isobutanol in water was prepared. Two grams ofthis solution were thoroughly mixed with 1.5 g of 3 Angstrom molecularsieve particles in a vial. After mixing, the vial was left to settle.Three phases were observed—a solid phase containing the molecular sieveparticles and two liquid phases comprising an upper, light,isobutanol-rich phase and a lower, heavy, isobutanol-lean phase.

What is claimed is:
 1. A method for recovering isobutanol from afermentation medium, comprising: (a) culturing a genetically engineeredmicroorganism in a fermentation medium comprising water and a feedstockcomprising isobutanol, said genetically engineered microorganism, and afermentable carbon source; (b) extracting the isobutanol in a portion ofthe fermentation medium with a water immiscible, isobutanol-selectiveliquid extractant, thereby forming an isobutanol-rich liquid phase and awater-rich liquid phase; (c) separating the isobutanol-rich liquid phaseand a water-rich liquid phase; (d) recovering the isobutanol from theisobutanol-rich liquid phase; wherein: said genetically engineeredmicroorganism is engineered to express or over-express a metabolicpathway that converts pyruvate to isobutanol; and said metabolic pathwaycomprises an acetohydroxy acid synthase (ALS) enzyme; and saidgenetically engineered microorganism includes a deletion of pyruvatedecarboxylase.
 2. The method of claim 1, wherein theisobutanol-selective liquid extractant is a hydrophobic hydrocarbonsolvent.
 3. The method of claim 2, wherein the hydrophobic hydrocarbonsolvent is a biofuel.
 4. The method of claim 1, wherein the ratio ofisobutanol to water in the isobutanol-rich phase is greater than about2.
 5. The method of claim 1, wherein the ratio of isobutanol to water inthe isobutanol-rich phase is greater than about
 8. 6. The method ofclaim 1, wherein the ratio of isobutanol to water in the isobutanol richphase is greater than the ratio of isobutanol to water in thefermentation medium by at least 5 fold.
 7. The method of claim 1,wherein the ratio of isobutanol to water in the isobutanol rich phase isgreater than the ratio of isobutanol to water in the fermentation mediumby at least 25-fold.
 8. The method of claim 1, further comprisingcooling the isobutanol-rich phase, thereby increasing the ratio ofisobutanol to water in the isobutanol-rich phase.
 9. The method of claim1, wherein the fermentation medium further comprises ethanol.
 10. Themethod of claim 1, further comprising: (e) conducting the water-richliquid phase to the fermentation medium.
 11. The method of claim 1,wherein the metabolic pathway comprises ketolacid reductoisomerase(KARI) and isobutyraldehyde dehydrogenase (IDH) enzymes.
 12. The methodof claim 11, wherein the KARI and IDH enzymes utilize a NADPH cofactor.13. The method of claim 11, wherein the KARI and IDH enzymes utilize aNADH cofactor.
 14. The method of claim 11, wherein either KARI or IDHutilizes a NADH cofactor.